Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-17 Thread Arnon Lavie

To add to this discussion:

Many of the comments to my question that started this thread do not 
sufficiently differentiate between accuracy and precision.
While we all want an assay that is internally consistent (i.e., high 
precision), we do care a lot about accuracy (the degree of closeness of 
measurements of a quantity to that quantity's actual (true) value [from 
http://en.wikipedia.org/wiki/Accuracy_and_precision])


One person actually stated that accuracy is not that important, but 
rather that precision is important. I disagree. When it comes to 
calculating kinetic parameters or binding spectrometry, an accurate 
reading (i.e. true) of the protein concentration is paramount.


So what I have been hearing (corrections welcome) is the following;
1. The Nanodrop may have an issue with precision due to various factors, 
such as evaporation while on the pedestal, dirt on the pedestal, and 
drifting from calibration. But if you squint, one can be happy with the 
machine


2. Bradford also as issues (low correlation with dye binding between 
standard protein used for calibration and one's sample). However, this 
assay can be highly precise.


There were only few comments on the NanoPhotometer™ Pearl, so more 
real-life experiences on that instrument would be helpful.


Cheers.

Arnon


--
***
Arnon Lavie, Professor
Dept. of Biochemistry and Molecular Genetics
University of Illinois at Chicago
900 S. Ashland Ave.
Molecular Biology Research Building, Room 1108 (M/C 669)
Chicago, IL 60607
U.S.A.
 Tel:(312) 355-5029
 Fax:(312) 355-4535
 E-mail: la...@uic.edu
 http://www.uic.edu/labs/lavie/
***


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Filip Van Petegem
Dear Arnon,

the Bradford method is not recommended for accurate measurements.  The
readings are strongly dependent on the amino acid composition.  A much
better method is using the absorption at 280nm under denaturing conditions
(6M Guanidine), and using calculated extinction coefficients based on the
composition of mostly Tyrosine and Tryptophan residues (+ disulfide bonds).
 This method is also old (Edelhoch, 1967), but very reliable.

One thing about the nanodrop: smaller volume = more evaporation.  On the
demo we've had, I was so unimpressed with the precision (25% variability
between two consecutive measurement) that we didn't consider this instrument
at all.  So unless you just want a 'rough' estimate, I wouldn't recommend it
at all. But most respectable spectrophotometers will take cuvettes with 50ul
volumes - a big step up from 1ml volumes...

Filip Van Petegem




On Thu, Jun 16, 2011 at 12:15 PM, Arnon Lavie la...@uic.edu wrote:

 Dear fellow crystallographers - a question about spectrophotometers for
 protein concentration determination.

 We are so last millennium - using Bradford reagent/ 1 ml cuvette for
 protein conc. determination.

 We have been considering buying a Nanodrop machine (small volume, no
 dilution needed, fast, easy).
 However, while testing our samples using a colleague's machine, we have
 gotten readings up to 100% different to our Bradford assay (all fully
 purified proteins). For example, Bradford says 6 mg/ml, Nanodrop 3 mg/ml. So
 while it is fun/easy to use the Nanodrop, I am not sure how reliable are the
 measurements (your thoughts?).

 So QUESTION 1: What are people's experience regarding the correlation
 between Nanodrop and Bradford?

 While researching the Nanodrop machine, I heard about the Implen
 NanoPhotmeter Pearl.
 So Question 2: Is the Pearl better/worse/same as the Nanodrop for our
 purpose?

 Thank you for helping us to advance to the next millennium, even if it is
 nearly a dozen years late.

 Arnon

 --
 ***
 Arnon Lavie, Professor
 Dept. of Biochemistry and Molecular Genetics
 University of Illinois at Chicago
 900 S. Ashland Ave.
 Molecular Biology Research Building, Room 1108 (M/C 669)
 Chicago, IL 60607
 U.S.A.
 Tel:(312) 355-5029
 Fax:(312) 355-4535
 E-mail: la...@uic.edu
 http://www.uic.edu/labs/lavie/
 ***




-- 
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: filip.vanpete...@gmail.com
http://crg.ubc.ca/VanPetegem/


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Oganesyan, Vaheh
I completely disagree with Filip's assessment. I've been using nanodrop nearly 
5 years and never had inconsistency issues. If you work at reasonable speed (if 
you put a drop there then lower the lever and click measure before you do 
anything else) there will be no issues. At very high concentrations the 
accuracy and therefore consistency may become lower. Concentrations between 5 
and 10 mg/ml should be fine. The instrument is pricey though.

 Vaheh




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Filip Van 
Petegem
Sent: Thursday, June 16, 2011 3:34 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old 
Bradford.

Dear Arnon,

the Bradford method is not recommended for accurate measurements.  The readings 
are strongly dependent on the amino acid composition.  A much better method is 
using the absorption at 280nm under denaturing conditions (6M Guanidine), and 
using calculated extinction coefficients based on the composition of mostly 
Tyrosine and Tryptophan residues (+ disulfide bonds).  This method is also old 
(Edelhoch, 1967), but very reliable.

One thing about the nanodrop: smaller volume = more evaporation.  On the demo 
we've had, I was so unimpressed with the precision (25% variability between 
two consecutive measurement) that we didn't consider this instrument at all.  
So unless you just want a 'rough' estimate, I wouldn't recommend it at all. But 
most respectable spectrophotometers will take cuvettes with 50ul volumes - a 
big step up from 1ml volumes...

Filip Van Petegem



On Thu, Jun 16, 2011 at 12:15 PM, Arnon Lavie 
la...@uic.edumailto:la...@uic.edu wrote:
Dear fellow crystallographers - a question about spectrophotometers for protein 
concentration determination.

We are so last millennium - using Bradford reagent/ 1 ml cuvette for protein 
conc. determination.

We have been considering buying a Nanodrop machine (small volume, no dilution 
needed, fast, easy).
However, while testing our samples using a colleague's machine, we have gotten 
readings up to 100% different to our Bradford assay (all fully purified 
proteins). For example, Bradford says 6 mg/ml, Nanodrop 3 mg/ml. So while it is 
fun/easy to use the Nanodrop, I am not sure how reliable are the measurements 
(your thoughts?).

So QUESTION 1: What are people's experience regarding the correlation between 
Nanodrop and Bradford?

While researching the Nanodrop machine, I heard about the Implen NanoPhotmeter 
Pearl.
So Question 2: Is the Pearl better/worse/same as the Nanodrop for our purpose?

Thank you for helping us to advance to the next millennium, even if it is 
nearly a dozen years late.

Arnon

--
***
Arnon Lavie, Professor
Dept. of Biochemistry and Molecular Genetics
University of Illinois at Chicago
900 S. Ashland Ave.
Molecular Biology Research Building, Room 1108 (M/C 669)
Chicago, IL 60607
U.S.A.
Tel:(312) 355-5029tel:%28312%29%20355-5029
Fax:(312) 355-4535tel:%28312%29%20355-4535
E-mail: la...@uic.edumailto:la...@uic.edu
http://www.uic.edu/labs/lavie/
***



--
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: filip.vanpete...@gmail.commailto:filip.vanpete...@gmail.com
http://crg.ubc.ca/VanPetegem/
To the extent this electronic communication or any of its attachments contain 
information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary. This communication is expected to 
be read and/or used only by the individual(s) for whom it is intended. If you 
have received this electronic communication in error, please reply to the 
sender advising of the error in transmission and delete the original message 
and any accompanying documents from your system immediately, without copying, 
reviewing or otherwise using them for any purpose. Thank you for your 
cooperation.


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread aaleshin
I also like our Nanodrop, but I do not recommend using it for Bradford 
measurements.

The 25% accuracy mentioned by Flip is pretty good for biological samples.  
Using 50 ul cuvette in a traditional spectrophotometer will not give this 
accuracy because cleanness of the cuvette will be a big issue...

Alex

On Jun 16, 2011, at 12:43 PM, Oganesyan, Vaheh wrote:

 I completely disagree with Filip’s assessment. I’ve been using nanodrop 
 nearly 5 years and never had inconsistency issues. If you work at reasonable 
 speed (if you put a drop there then lower the lever and click measure before 
 you do anything else) there will be no issues. At very high concentrations 
 the accuracy and therefore consistency may become lower. Concentrations 
 between 5 and 10 mg/ml should be fine. The instrument is pricey though.
  
  Vaheh 
  
  
  
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Filip 
 Van Petegem
 Sent: Thursday, June 16, 2011 3:34 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old 
 Bradford.
  
 Dear Arnon,
  
 the Bradford method is not recommended for accurate measurements.  The 
 readings are strongly dependent on the amino acid composition.  A much better 
 method is using the absorption at 280nm under denaturing conditions (6M 
 Guanidine), and using calculated extinction coefficients based on the 
 composition of mostly Tyrosine and Tryptophan residues (+ disulfide bonds).  
 This method is also old (Edelhoch, 1967), but very reliable.
  
 One thing about the nanodrop: smaller volume = more evaporation.  On the demo 
 we've had, I was so unimpressed with the precision (25% variability between 
 two consecutive measurement) that we didn't consider this instrument at all.  
 So unless you just want a 'rough' estimate, I wouldn't recommend it at all. 
 But most respectable spectrophotometers will take cuvettes with 50ul volumes 
 - a big step up from 1ml volumes...
  
 Filip Van Petegem
  
  
   
 On Thu, Jun 16, 2011 at 12:15 PM, Arnon Lavie la...@uic.edu wrote:
 Dear fellow crystallographers - a question about spectrophotometers for 
 protein concentration determination.
 
 We are so last millennium - using Bradford reagent/ 1 ml cuvette for protein 
 conc. determination.
 
 We have been considering buying a Nanodrop machine (small volume, no dilution 
 needed, fast, easy).
 However, while testing our samples using a colleague's machine, we have 
 gotten readings up to 100% different to our Bradford assay (all fully 
 purified proteins). For example, Bradford says 6 mg/ml, Nanodrop 3 mg/ml. So 
 while it is fun/easy to use the Nanodrop, I am not sure how reliable are the 
 measurements (your thoughts?).
 
 So QUESTION 1: What are people's experience regarding the correlation between 
 Nanodrop and Bradford?
 
 While researching the Nanodrop machine, I heard about the Implen 
 NanoPhotmeter Pearl.
 So Question 2: Is the Pearl better/worse/same as the Nanodrop for our purpose?
 
 Thank you for helping us to advance to the next millennium, even if it is 
 nearly a dozen years late.
 
 Arnon
 
 -- 
 ***
 Arnon Lavie, Professor
 Dept. of Biochemistry and Molecular Genetics
 University of Illinois at Chicago
 900 S. Ashland Ave.
 Molecular Biology Research Building, Room 1108 (M/C 669)
 Chicago, IL 60607
 U.S.A.
 Tel:(312) 355-5029
 Fax:(312) 355-4535
 E-mail: la...@uic.edu
 http://www.uic.edu/labs/lavie/
 ***
 
 
 
 -- 
 Filip Van Petegem, PhD
 Assistant Professor
 The University of British Columbia
 Dept. of Biochemistry and Molecular Biology
 2350 Health Sciences Mall - Rm 2.356
 Vancouver, V6T 1Z3
 
 phone: +1 604 827 4267
 email: filip.vanpete...@gmail.com
 http://crg.ubc.ca/VanPetegem/
 To the extent this electronic communication or any of its attachments contain 
 information that is not in the public domain, such information is considered 
 by MedImmune to be confidential and proprietary. This communication is 
 expected to be read and/or used only by the individual(s) for whom it is 
 intended. If you have received this electronic communication in error, please 
 reply to the sender advising of the error in transmission and delete the 
 original message and any accompanying documents from your system immediately, 
 without copying, reviewing or otherwise using them for any purpose. Thank you 
 for your cooperation.



Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Francis E Reyes
Never had problems with evaporation (and this is in the relatively dry  
climate of Denver, CO, especially in the winter when the relative  
humidity is in the low 20%).


Using the Thermo Scientific Nanodrop 2000c.

We use it also as a prerequisite for ITC, which can be very sensitive  
to proper concentrations.


F


On Jun 16, 2011, at 1:15 PM, Arnon Lavie wrote:

Dear fellow crystallographers - a question about spectrophotometers  
for protein concentration determination.


We are so last millennium - using Bradford reagent/ 1 ml cuvette for  
protein conc. determination.


We have been considering buying a Nanodrop machine (small volume, no  
dilution needed, fast, easy).
However, while testing our samples using a colleague's machine, we  
have gotten readings up to 100% different to our Bradford assay (all  
fully purified proteins). For example, Bradford says 6 mg/ml,  
Nanodrop 3 mg/ml. So while it is fun/easy to use the Nanodrop, I am  
not sure how reliable are the measurements (your thoughts?).


So QUESTION 1: What are people's experience regarding the  
correlation between Nanodrop and Bradford?


While researching the Nanodrop machine, I heard about the Implen  
NanoPhotmeter Pearl.
So Question 2: Is the Pearl better/worse/same as the Nanodrop for  
our purpose?


Thank you for helping us to advance to the next millennium, even if  
it is nearly a dozen years late.


Arnon

--
***
Arnon Lavie, Professor
Dept. of Biochemistry and Molecular Genetics
University of Illinois at Chicago
900 S. Ashland Ave.
Molecular Biology Research Building, Room 1108 (M/C 669)
Chicago, IL 60607
U.S.A.
Tel:(312) 355-5029
Fax:(312) 355-4535
E-mail: la...@uic.edu
http://www.uic.edu/labs/lavie/
***


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Bosch, Juergen
I second Vaheh,

been using Nanodrops at three different locations and have been happy with them 
plus reproducibility of results. If you have 50 mg/ml you'll need to dilute, 
but we rarely get that high anyhow.

Additionally you safe time. If you had a cuvette based system, you should 
always clean before and after yourself and the time it takes to do that is 
wasted. With the Nanodrop simply use a Kim wipe and EtOH before and H2O after 
your protein followed by EtOH.

Jürgen

On Jun 16, 2011, at 3:43 PM, Oganesyan, Vaheh wrote:

I completely disagree with Filip’s assessment. I’ve been using nanodrop nearly 
5 years and never had inconsistency issues. If you work at reasonable speed (if 
you put a drop there then lower the lever and click measure before you do 
anything else) there will be no issues. At very high concentrations the 
accuracy and therefore consistency may become lower. Concentrations between 5 
and 10 mg/ml should be fine. The instrument is pricey though.

 Vaheh




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Filip Van 
Petegem
Sent: Thursday, June 16, 2011 3:34 PM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old 
Bradford.

Dear Arnon,

the Bradford method is not recommended for accurate measurements.  The readings 
are strongly dependent on the amino acid composition.  A much better method is 
using the absorption at 280nm under denaturing conditions (6M Guanidine), and 
using calculated extinction coefficients based on the composition of mostly 
Tyrosine and Tryptophan residues (+ disulfide bonds).  This method is also old 
(Edelhoch, 1967), but very reliable.

One thing about the nanodrop: smaller volume = more evaporation.  On the demo 
we've had, I was so unimpressed with the precision (25% variability between 
two consecutive measurement) that we didn't consider this instrument at all.  
So unless you just want a 'rough' estimate, I wouldn't recommend it at all. But 
most respectable spectrophotometers will take cuvettes with 50ul volumes - a 
big step up from 1ml volumes...

Filip Van Petegem



On Thu, Jun 16, 2011 at 12:15 PM, Arnon Lavie 
la...@uic.edumailto:la...@uic.edu wrote:
Dear fellow crystallographers - a question about spectrophotometers for protein 
concentration determination.

We are so last millennium - using Bradford reagent/ 1 ml cuvette for protein 
conc. determination.

We have been considering buying a Nanodrop machine (small volume, no dilution 
needed, fast, easy).
However, while testing our samples using a colleague's machine, we have gotten 
readings up to 100% different to our Bradford assay (all fully purified 
proteins). For example, Bradford says 6 mg/ml, Nanodrop 3 mg/ml. So while it is 
fun/easy to use the Nanodrop, I am not sure how reliable are the measurements 
(your thoughts?).

So QUESTION 1: What are people's experience regarding the correlation between 
Nanodrop and Bradford?

While researching the Nanodrop machine, I heard about the Implen NanoPhotmeter 
Pearl.
So Question 2: Is the Pearl better/worse/same as the Nanodrop for our purpose?

Thank you for helping us to advance to the next millennium, even if it is 
nearly a dozen years late.

Arnon

--
***
Arnon Lavie, Professor
Dept. of Biochemistry and Molecular Genetics
University of Illinois at Chicago
900 S. Ashland Ave.
Molecular Biology Research Building, Room 1108 (M/C 669)
Chicago, IL 60607
U.S.A.
Tel:(312) 355-5029tel:%28312%29%20355-5029
Fax:(312) 355-4535tel:%28312%29%20355-4535
E-mail: la...@uic.edumailto:la...@uic.edu
http://www.uic.edu/labs/lavie/
***



--
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: filip.vanpete...@gmail.commailto:filip.vanpete...@gmail.com
http://crg.ubc.ca/VanPetegem/
To the extent this electronic communication or any of its attachments contain 
information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary. This communication is expected to 
be read and/or used only by the individual(s) for whom it is intended. If you 
have received this electronic communication in error, please reply to the 
sender advising of the error in transmission and delete the original message 
and any accompanying documents from your system immediately, without copying, 
reviewing or otherwise using them for any purpose. Thank you for your 
cooperation.

..
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology

Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Filip Van Petegem
25% is not acceptable for ITC or CD experiments though...

I was just sharing our bad experience with a demo nanodrop we had. Even if
evaporation is not an issue, one has to take pipetting errors into account
when dealing with small volumes.  The relative error on 1-2ul is a lot
bigger than on 50ul. Unless you want to pre-mix 50ul and use a small
quantity of that, which defeats the purpose of miniaturization...  It all
depends on your applications and sample availability, but if you want a very
accurate measurement, miniaturized volumes just won't get you the same
accuracy.

Cuvettes will give a better accuracy provided you clean them properly. Just
some water or EtOH is *not* enough...

Filip Van Petegem



On Thu, Jun 16, 2011 at 12:52 PM, aaleshin aales...@burnham.org wrote:

 I also like our Nanodrop, but I do not recommend using it for Bradford
 measurements.

 The 25% accuracy mentioned by Flip is pretty good for biological samples.
  Using 50 ul cuvette in a traditional spectrophotometer will not give this
 accuracy because cleanness of the cuvette will be a big issue...

 Alex

 On Jun 16, 2011, at 12:43 PM, Oganesyan, Vaheh wrote:

 I completely disagree with Filip’s assessment. I’ve been using nanodrop
 nearly 5 years and never had inconsistency issues. If you work at reasonable
 speed (if you put a drop there then lower the lever and click measure before
 you do anything else) there will be no issues. At very high concentrations
 the accuracy and therefore consistency may become lower. Concentrations
 between 5 and 10 mg/ml should be fine. The instrument is pricey though.


 * Vaheh*






 --
 *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
 *Filip
 Van Petegem
 *Sent:* Thursday, June 16, 2011 3:34 PM
 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good
 old Bradford.


 Dear Arnon,


 the Bradford method is not recommended for accurate measurements.  The
 readings are strongly dependent on the amino acid composition.  A much
 better method is using the absorption at 280nm under denaturing conditions
 (6M Guanidine), and using calculated extinction coefficients based on the
 composition of mostly Tyrosine and Tryptophan residues (+ disulfide bonds).
  This method is also old (Edelhoch, 1967), but very reliable.


 One thing about the nanodrop: smaller volume = more evaporation.  On the
 demo we've had, I was so unimpressed with the precision (25% variability
 between two consecutive measurement) that we didn't consider this instrument
 at all.  So unless you just want a 'rough' estimate, I wouldn't recommend it
 at all. But most respectable spectrophotometers will take cuvettes with 50ul
 volumes - a big step up from 1ml volumes...


 Filip Van Petegem





 On Thu, Jun 16, 2011 at 12:15 PM, Arnon Lavie la...@uic.edu wrote:
 Dear fellow crystallographers - a question about spectrophotometers for
 protein concentration determination.

 We are so last millennium - using Bradford reagent/ 1 ml cuvette for
 protein conc. determination.

 We have been considering buying a Nanodrop machine (small volume, no
 dilution needed, fast, easy).
 However, while testing our samples using a colleague's machine, we have
 gotten readings up to 100% different to our Bradford assay (all fully
 purified proteins). For example, Bradford says 6 mg/ml, Nanodrop 3 mg/ml. So
 while it is fun/easy to use the Nanodrop, I am not sure how reliable are the
 measurements (your thoughts?).

 So QUESTION 1: What are people's experience regarding the correlation
 between Nanodrop and Bradford?

 While researching the Nanodrop machine, I heard about the Implen
 NanoPhotmeter Pearl.
 So Question 2: Is the Pearl better/worse/same as the Nanodrop for our
 purpose?

 Thank you for helping us to advance to the next millennium, even if it is
 nearly a dozen years late.

 Arnon

 --
 ***
 Arnon Lavie, Professor
 Dept. of Biochemistry and Molecular Genetics
 University of Illinois at Chicago
 900 S. Ashland Ave.
 Molecular Biology Research Building, Room 1108 (M/C 669)
 Chicago, IL 60607
 U.S.A.
 Tel:(312) 355-5029
 Fax:(312) 355-4535
 E-mail: la...@uic.edu
 http://www.uic.edu/labs/lavie/
 ***



 --
 Filip Van Petegem, PhD
 Assistant Professor
 The University of British Columbia
 Dept. of Biochemistry and Molecular Biology
 2350 Health Sciences Mall - Rm 2.356
 Vancouver, V6T 1Z3

 phone: +1 604 827 4267
 email: filip.vanpete...@gmail.com
 http://crg.ubc.ca/VanPetegem/
 To the extent this electronic communication or any of its attachments
 contain information that is not in the public domain, such information is
 considered by MedImmune to be confidential and proprietary. This
 communication

Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Quyen Hoang

We also have not experienced any problems with a Nanodrop 2000C.
No one in my touched the two boxes of Bradford and BCA kits that we  
have, because we have been very happy with the Nanodrop.


Quyen
___
Quyen Hoang, Ph.D
Assistant Professor
Department of Biochemistry and Molecular Biology,
Stark Neurosciences Research Institute
Indiana University School of Medicine
635 Barnhill Drive, Room MS0013D
Indianapolis, Indiana 46202-5122

Phone: 317-274-4371
Fax: 317-274-4686
email: qqho...@iupui.edu
Website: www.hoanglab.com



On Jun 16, 2011, at 3:54 PM, Francis E Reyes wrote:

Never had problems with evaporation (and this is in the relatively  
dry climate of Denver, CO, especially in the winter when the  
relative humidity is in the low 20%).


Using the Thermo Scientific Nanodrop 2000c.

We use it also as a prerequisite for ITC, which can be very  
sensitive to proper concentrations.


F


On Jun 16, 2011, at 1:15 PM, Arnon Lavie wrote:

Dear fellow crystallographers - a question about spectrophotometers  
for protein concentration determination.


We are so last millennium - using Bradford reagent/ 1 ml cuvette  
for protein conc. determination.


We have been considering buying a Nanodrop machine (small volume,  
no dilution needed, fast, easy).
However, while testing our samples using a colleague's machine, we  
have gotten readings up to 100% different to our Bradford assay  
(all fully purified proteins). For example, Bradford says 6 mg/ml,  
Nanodrop 3 mg/ml. So while it is fun/easy to use the Nanodrop, I am  
not sure how reliable are the measurements (your thoughts?).


So QUESTION 1: What are people's experience regarding the  
correlation between Nanodrop and Bradford?


While researching the Nanodrop machine, I heard about the Implen  
NanoPhotmeter Pearl.
So Question 2: Is the Pearl better/worse/same as the Nanodrop for  
our purpose?


Thank you for helping us to advance to the next millennium, even if  
it is nearly a dozen years late.


Arnon

--
***
Arnon Lavie, Professor
Dept. of Biochemistry and Molecular Genetics
University of Illinois at Chicago
900 S. Ashland Ave.
Molecular Biology Research Building, Room 1108 (M/C 669)
Chicago, IL 60607
U.S.A.
   Tel:(312) 355-5029
   Fax:(312) 355-4535
   E-mail: la...@uic.edu
   http://www.uic.edu/labs/lavie/
***




Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Bjørn Panyella Pedersen

On 2011-06-16 13:06, Filip Van Petegem wrote:

Even if evaporation is not an issue, one has to take pipetting errors into 
account
when dealing with small volumes.  The relative error on 1-2ul is a lot bigger 
than on 50ul.


True, but the nanodrop works independent of volumes, since it has a 
fixed pathlength.  1ul loaded will give the same result as 2ul loaded.


hth
-Bjørn
--
Bjørn Panyella Pedersen
Macromolecular Structure Group
Dept. of Biochemistry and Biophysics
University of California, San Francisco


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread aaleshin
Filip,
25% accuracy is observed only for very diluted (OD280 0.1) or concentrated 
samples. But those sample a rarely used for ITC or CD. The concentrated samples 
require dilution but a regular spec does it too. Since the light passway is 
very short in Nanodrop it is accurate with more concentrated samples, which we 
crystallographers use, so Nanodrop is ideal instrument for our trade.

If the drop is within recommended volume like 1-2 ul for our model, its size 
has a very small influence on the measurement. 

 Cuvettes will give a better accuracy provided you clean them properly. 
I hated those times when I had to measure a concentration because of a need to 
wash a cuvette. In a biological lab they are always dirty. We switched to 
plastic disposable cuvettes for that reason...

Alex

On Jun 16, 2011, at 1:06 PM, Filip Van Petegem wrote:

 25% is not acceptable for ITC or CD experiments though...
 
 I was just sharing our bad experience with a demo nanodrop we had. Even if 
 evaporation is not an issue, one has to take pipetting errors into account 
 when dealing with small volumes.  The relative error on 1-2ul is a lot bigger 
 than on 50ul. Unless you want to pre-mix 50ul and use a small quantity of 
 that, which defeats the purpose of miniaturization...  It all depends on your 
 applications and sample availability, but if you want a very accurate 
 measurement, miniaturized volumes just won't get you the same accuracy.
 
 Cuvettes will give a better accuracy provided you clean them properly. Just 
 some water or EtOH is *not* enough...
 
 Filip Van Petegem
 
 
 
 On Thu, Jun 16, 2011 at 12:52 PM, aaleshin aales...@burnham.org wrote:
 I also like our Nanodrop, but I do not recommend using it for Bradford 
 measurements.
 
 The 25% accuracy mentioned by Flip is pretty good for biological samples.  
 Using 50 ul cuvette in a traditional spectrophotometer will not give this 
 accuracy because cleanness of the cuvette will be a big issue...
 
 Alex
 
 On Jun 16, 2011, at 12:43 PM, Oganesyan, Vaheh wrote:
 
 I completely disagree with Filip’s assessment. I’ve been using nanodrop 
 nearly 5 years and never had inconsistency issues. If you work at reasonable 
 speed (if you put a drop there then lower the lever and click measure before 
 you do anything else) there will be no issues. At very high concentrations 
 the accuracy and therefore consistency may become lower. Concentrations 
 between 5 and 10 mg/ml should be fine. The instrument is pricey though.
  
  Vaheh 
  
  
  
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Filip 
 Van Petegem
 Sent: Thursday, June 16, 2011 3:34 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old 
 Bradford.
  
 Dear Arnon,
  
 the Bradford method is not recommended for accurate measurements.  The 
 readings are strongly dependent on the amino acid composition.  A much 
 better method is using the absorption at 280nm under denaturing conditions 
 (6M Guanidine), and using calculated extinction coefficients based on the 
 composition of mostly Tyrosine and Tryptophan residues (+ disulfide bonds).  
 This method is also old (Edelhoch, 1967), but very reliable.
  
 One thing about the nanodrop: smaller volume = more evaporation.  On the 
 demo we've had, I was so unimpressed with the precision (25% variability 
 between two consecutive measurement) that we didn't consider this instrument 
 at all.  So unless you just want a 'rough' estimate, I wouldn't recommend it 
 at all. But most respectable spectrophotometers will take cuvettes with 50ul 
 volumes - a big step up from 1ml volumes...
  
 Filip Van Petegem
  
  
   
 On Thu, Jun 16, 2011 at 12:15 PM, Arnon Lavie la...@uic.edu wrote:
 Dear fellow crystallographers - a question about spectrophotometers for 
 protein concentration determination.
 
 We are so last millennium - using Bradford reagent/ 1 ml cuvette for protein 
 conc. determination.
 
 We have been considering buying a Nanodrop machine (small volume, no 
 dilution needed, fast, easy).
 However, while testing our samples using a colleague's machine, we have 
 gotten readings up to 100% different to our Bradford assay (all fully 
 purified proteins). For example, Bradford says 6 mg/ml, Nanodrop 3 mg/ml. So 
 while it is fun/easy to use the Nanodrop, I am not sure how reliable are the 
 measurements (your thoughts?).
 
 So QUESTION 1: What are people's experience regarding the correlation 
 between Nanodrop and Bradford?
 
 While researching the Nanodrop machine, I heard about the Implen 
 NanoPhotmeter Pearl.
 So Question 2: Is the Pearl better/worse/same as the Nanodrop for our 
 purpose?
 
 Thank you for helping us to advance to the next millennium, even if it is 
 nearly a dozen years late.
 
 Arnon
 
 -- 
 ***
 Arnon Lavie, Professor
 Dept. of Biochemistry and Molecular Genetics
 University of Illinois at Chicago
 900 S

Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread David Briggs
I'll give my backing to the Nanodrop as well.

I've used it in two different labs, for general yield checking use as
well as prior to ITC experiments, and haven't found there to be any
issues.

That said, I've also used cuvettes, and I find that one the whole,
cuvette-derived and nanodrop-derived measurements are comparable.

I wouldn't touch Bradford with a barge-pole. I've found it to be
wildly inaccurate for certain proteins I've handled, where as the
OD280 measurements have been fine.

Dave


David C. Briggs PhD
Father, Structural Biologist and Sceptic

University of Manchester E-mail:
david.c.bri...@manchester.ac.uk

http://manchester.academia.edu/DavidBriggs (v.sensible)
http://xtaldave.wordpress.com/ (sensible)
http://xtaldave.posterous.com/ (less sensible)
Twitter: @xtaldave
Skype: DocDCB




On 16 June 2011 21:15, Quyen Hoang qqho...@gmail.com wrote:
 We also have not experienced any problems with a Nanodrop 2000C.
 No one in my touched the two boxes of Bradford and BCA kits that we have,
 because we have been very happy with the Nanodrop.
 Quyen
 ___
 Quyen Hoang, Ph.D
 Assistant Professor
 Department of Biochemistry and Molecular Biology,
 Stark Neurosciences Research Institute
 Indiana University School of Medicine
 635 Barnhill Drive, Room MS0013D
 Indianapolis, Indiana 46202-5122
 Phone: 317-274-4371
 Fax: 317-274-4686
 email: qqho...@iupui.edu
 Website: www.hoanglab.com


 On Jun 16, 2011, at 3:54 PM, Francis E Reyes wrote:

 Never had problems with evaporation (and this is in the relatively dry
 climate of Denver, CO, especially in the winter when the relative humidity
 is in the low 20%).

 Using the Thermo Scientific Nanodrop 2000c.

 We use it also as a prerequisite for ITC, which can be very sensitive to
 proper concentrations.

 F


 On Jun 16, 2011, at 1:15 PM, Arnon Lavie wrote:

 Dear fellow crystallographers - a question about spectrophotometers for
 protein concentration determination.

 We are so last millennium - using Bradford reagent/ 1 ml cuvette for protein
 conc. determination.

 We have been considering buying a Nanodrop machine (small volume, no
 dilution needed, fast, easy).

 However, while testing our samples using a colleague's machine, we have
 gotten readings up to 100% different to our Bradford assay (all fully
 purified proteins). For example, Bradford says 6 mg/ml, Nanodrop 3 mg/ml. So
 while it is fun/easy to use the Nanodrop, I am not sure how reliable are the
 measurements (your thoughts?).

 So QUESTION 1: What are people's experience regarding the correlation
 between Nanodrop and Bradford?

 While researching the Nanodrop machine, I heard about the Implen
 NanoPhotmeter Pearl.

 So Question 2: Is the Pearl better/worse/same as the Nanodrop for our
 purpose?

 Thank you for helping us to advance to the next millennium, even if it is
 nearly a dozen years late.

 Arnon

 --

 ***

 Arnon Lavie, Professor

 Dept. of Biochemistry and Molecular Genetics

 University of Illinois at Chicago

 900 S. Ashland Ave.

 Molecular Biology Research Building, Room 1108 (M/C 669)

 Chicago, IL 60607

 U.S.A.

    Tel:    (312) 355-5029

    Fax:    (312) 355-4535

    E-mail: la...@uic.edu

    http://www.uic.edu/labs/lavie/

 ***




Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Tommi Kajander
I would add that there are some issues with air, you have to be careful with 
nanodrop that the path is ok, and also if concentrations are low, 1 mg/ml for 
instance, i am not sure one can trust it - compare 50 ul 1 cm path results with 
nano at 0.5-1 mg/ml... i get inconsistency there.. its good for concentrated 
samples and fast. and handy, we use it a lot, but
for dilute samples, i always use 1 cm light path. just my feel on it.

Tommi

On Jun 16, 2011, at 11:20 PM, aaleshin wrote:

 Filip,
 25% accuracy is observed only for very diluted (OD280 0.1) or concentrated 
 samples. But those sample a rarely used for ITC or CD. The concentrated 
 samples require dilution but a regular spec does it too. Since the light 
 passway is very short in Nanodrop it is accurate with more concentrated 
 samples, which we crystallographers use, so Nanodrop is ideal instrument for 
 our trade.
 
 If the drop is within recommended volume like 1-2 ul for our model, its size 
 has a very small influence on the measurement. 
 
 Cuvettes will give a better accuracy provided you clean them properly. 
 I hated those times when I had to measure a concentration because of a need 
 to wash a cuvette. In a biological lab they are always dirty. We switched to 
 plastic disposable cuvettes for that reason...
 
 Alex
 
 On Jun 16, 2011, at 1:06 PM, Filip Van Petegem wrote:
 
 25% is not acceptable for ITC or CD experiments though...
 
 I was just sharing our bad experience with a demo nanodrop we had. Even if 
 evaporation is not an issue, one has to take pipetting errors into account 
 when dealing with small volumes.  The relative error on 1-2ul is a lot 
 bigger than on 50ul. Unless you want to pre-mix 50ul and use a small 
 quantity of that, which defeats the purpose of miniaturization...  It all 
 depends on your applications and sample availability, but if you want a very 
 accurate measurement, miniaturized volumes just won't get you the same 
 accuracy.
 
 Cuvettes will give a better accuracy provided you clean them properly. Just 
 some water or EtOH is *not* enough...
 
 Filip Van Petegem
 
 
 
 On Thu, Jun 16, 2011 at 12:52 PM, aaleshin aales...@burnham.org wrote:
 I also like our Nanodrop, but I do not recommend using it for Bradford 
 measurements.
 
 The 25% accuracy mentioned by Flip is pretty good for biological samples.  
 Using 50 ul cuvette in a traditional spectrophotometer will not give this 
 accuracy because cleanness of the cuvette will be a big issue...
 
 Alex
 
 On Jun 16, 2011, at 12:43 PM, Oganesyan, Vaheh wrote:
 
 I completely disagree with Filip’s assessment. I’ve been using nanodrop 
 nearly 5 years and never had inconsistency issues. If you work at 
 reasonable speed (if you put a drop there then lower the lever and click 
 measure before you do anything else) there will be no issues. At very high 
 concentrations the accuracy and therefore consistency may become lower. 
 Concentrations between 5 and 10 mg/ml should be fine. The instrument is 
 pricey though.
  
  Vaheh 
  
  
  
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Filip 
 Van Petegem
 Sent: Thursday, June 16, 2011 3:34 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old 
 Bradford.
  
 Dear Arnon,
  
 the Bradford method is not recommended for accurate measurements.  The 
 readings are strongly dependent on the amino acid composition.  A much 
 better method is using the absorption at 280nm under denaturing conditions 
 (6M Guanidine), and using calculated extinction coefficients based on the 
 composition of mostly Tyrosine and Tryptophan residues (+ disulfide bonds). 
  This method is also old (Edelhoch, 1967), but very reliable.
  
 One thing about the nanodrop: smaller volume = more evaporation.  On the 
 demo we've had, I was so unimpressed with the precision (25% variability 
 between two consecutive measurement) that we didn't consider this 
 instrument at all.  So unless you just want a 'rough' estimate, I wouldn't 
 recommend it at all. But most respectable spectrophotometers will take 
 cuvettes with 50ul volumes - a big step up from 1ml volumes...
  
 Filip Van Petegem
  
  
   
 On Thu, Jun 16, 2011 at 12:15 PM, Arnon Lavie la...@uic.edu wrote:
 Dear fellow crystallographers - a question about spectrophotometers for 
 protein concentration determination.
 
 We are so last millennium - using Bradford reagent/ 1 ml cuvette for 
 protein conc. determination.
 
 We have been considering buying a Nanodrop machine (small volume, no 
 dilution needed, fast, easy).
 However, while testing our samples using a colleague's machine, we have 
 gotten readings up to 100% different to our Bradford assay (all fully 
 purified proteins). For example, Bradford says 6 mg/ml, Nanodrop 3 mg/ml. 
 So while it is fun/easy to use the Nanodrop, I am not sure how reliable are 
 the measurements (your thoughts?).
 
 So QUESTION 1: What are people's experience regarding

Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Prince, D Bryan
Dear Arnon,

I have a Nanodrop2000, which reads from the post or a user supplied
cuvette. I have had NO complaints about using the Nanodrop for reading
protein concentration immediately prior to crystallization setup. When I
have observed differences in OD280 vs Bradford, it is usually due to one
of the following:

1) The protein is deficient in the amino acid residues that provide the
280nm signal. This can be corrected with the extinction coefficient, and
can be programmed into the Nanodrop so that the readout is correct.

2) The protein is behaving badly in the Bradford assay (interference
with some component of the protein buffer)

3) The dilution used in the Bradford is contributing to large
concentration errors (and can be combined with 2, above)

The value of the Nanodrop, is that you get a OD280 that you can
reproduce prior to every experiment at a low cost of protein sample. The
N2000 can also do the Bradford or other assay on the post, or with a
cuvette if you really want to do it that way. When there have been no
mitigating factors, my Nanodrop OD280 readings have been within 5% of
the values I get from SEC-MALS, MassSpec or Bradford. I have no
experience with the Pearl, but I am very happy with my Nanodrop 2000.

Good luck with your choice!

Bryan


--
Confidentiality Notice: This message is private and may contain confidential 
and proprietary information. If you have received this message in error, please 
notify us and remove it from your system and note that you must not copy, 
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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Arnon Lavie
Sent: Thursday, June 16, 2011 3:16 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old
Bradford.

Dear fellow crystallographers - a question about spectrophotometers for
protein concentration determination.

We are so last millennium - using Bradford reagent/ 1 ml cuvette for
protein conc. determination.

We have been considering buying a Nanodrop machine (small volume, no
dilution needed, fast, easy).
However, while testing our samples using a colleague's machine, we have
gotten readings up to 100% different to our Bradford assay (all fully
purified proteins). For example, Bradford says 6 mg/ml, Nanodrop 3
mg/ml. So while it is fun/easy to use the Nanodrop, I am not sure how
reliable are the measurements (your thoughts?).

So QUESTION 1: What are people's experience regarding the correlation
between Nanodrop and Bradford?

While researching the Nanodrop machine, I heard about the Implen
NanoPhotmeter Pearl.
So Question 2: Is the Pearl better/worse/same as the Nanodrop for our
purpose?

Thank you for helping us to advance to the next millennium, even if it
is nearly a dozen years late.

Arnon

--
***
Arnon Lavie, Professor
Dept. of Biochemistry and Molecular Genetics
University of Illinois at Chicago
900 S. Ashland Ave.
Molecular Biology Research Building, Room 1108 (M/C 669)
Chicago, IL 60607
U.S.A.
  Tel:(312) 355-5029
  Fax:(312) 355-4535
  E-mail: la...@uic.edu
  http://www.uic.edu/labs/lavie/
***


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Chun Luo
Although the path length of NanoDrop is fixed. 1 ul may not form good liquid
column. One trick is to spot a little bit more sample, 2-4 ul, on Nanodrop,
specially for concentrated protein samples with glycerol and E. coli
culture. It eliminate the bubble problem. 

To get good reading, a new drop need to be used for each measurement. The
liquid column may not form well when repeat with the same drop. Take a look
at the liquid column. Nanodrop actually measures the sample twice for each
reading. In consistent measurements give error message. As all the machines
with moving parts, periodical maintenance is needed to give good readings.

There may be a huge difference between Bradford and A200. In some cases none
of them gives the true concentration. I wouldn't abandon Bradford, which by
itself is very consistent if done right. Indicating how protein
concentration is measured in publications will help the research community.

Cheers,

Chun


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bjørn
Panyella Pedersen
Sent: Thursday, June 16, 2011 1:19 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old
Bradford.

On 2011-06-16 13:06, Filip Van Petegem wrote:
 Even if evaporation is not an issue, one has to take pipetting errors into
account
 when dealing with small volumes.  The relative error on 1-2ul is a lot
bigger than on 50ul.

True, but the nanodrop works independent of volumes, since it has a 
fixed pathlength.  1ul loaded will give the same result as 2ul loaded.

hth
-Bjørn
-- 
Bjørn Panyella Pedersen
Macromolecular Structure Group
Dept. of Biochemistry and Biophysics
University of California, San Francisco


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Justin Hall

Hi Alex,

I read Filip's comment about volume not as a path length argument, but  
about concentration uncertainty in mixing small volumes to dilute a  
sample down before measuring it (?). I have never had to make a  
dilution for my nanodrop (my proteins are usually not that  
concentrated), but I could see his point if I did have to.


As for the variance between samples, I don't know about 25%, but I  
have observed multiple readings to have variance. I always take 3  
readings on my nanodrop and then average them to deal with the  
variance I see. I don't mind doing this because the instrument is so  
fast, and I don't mind the cost at 6 ul of sample total.


The most variance I have seen is usually in spin columns, where I will  
be doing a buffer exchange from a storage buffer (sometimes at ca. 20%  
glycerol) into an assay or xstal buffer, and I have wondered to myself  
if the variance I see could be due to incomplete mixing of a protein  
sample betwen a viscous buffer at the bottom with the rest of the  
buffer. I don't know how often other people find themselves in a  
situation where they may be sampling their 2 ul from a  
micro-environment that is not homogenous with the rest of the  
sample, but with small volumes I think that be a problem. Food for  
thought.


Filip, I would buy a nanodrop. It is much better than a  
Bradford/cuvette and your students will love you for it. Cheers~


~Justin


Quoting aaleshin aales...@burnham.org:


Filip,
25% accuracy is observed only for very diluted (OD280 0.1) or  
concentrated samples. But those sample a rarely used for ITC or CD.  
The concentrated samples require dilution but a regular spec does it  
too. Since the light passway is very short in Nanodrop it is  
accurate with more concentrated samples, which we crystallographers  
use, so Nanodrop is ideal instrument for our trade.


If the drop is within recommended volume like 1-2 ul for our model,  
its size has a very small influence on the measurement.



Cuvettes will give a better accuracy provided you clean them properly.
I hated those times when I had to measure a concentration because of  
a need to wash a cuvette. In a biological lab they are always dirty.  
We switched to plastic disposable cuvettes for that reason...


Alex

On Jun 16, 2011, at 1:06 PM, Filip Van Petegem wrote:


25% is not acceptable for ITC or CD experiments though...

I was just sharing our bad experience with a demo nanodrop we had.  
Even if evaporation is not an issue, one has to take pipetting  
errors into account when dealing with small volumes.  The relative  
error on 1-2ul is a lot bigger than on 50ul. Unless you want to  
pre-mix 50ul and use a small quantity of that, which defeats the  
purpose of miniaturization...  It all depends on your applications  
and sample availability, but if you want a very accurate  
measurement, miniaturized volumes just won't get you the same  
accuracy.


Cuvettes will give a better accuracy provided you clean them  
properly. Just some water or EtOH is *not* enough...


Filip Van Petegem



On Thu, Jun 16, 2011 at 12:52 PM, aaleshin aales...@burnham.org wrote:
I also like our Nanodrop, but I do not recommend using it for  
Bradford measurements.


The 25% accuracy mentioned by Flip is pretty good for biological  
samples.  Using 50 ul cuvette in a traditional spectrophotometer  
will not give this accuracy because cleanness of the cuvette will  
be a big issue...


Alex

On Jun 16, 2011, at 12:43 PM, Oganesyan, Vaheh wrote:

I completely disagree with Filip’s assessment. I’ve been using  
nanodrop nearly 5 years and never had inconsistency issues. If you  
work at reasonable speed (if you put a drop there then lower the  
lever and click measure before you do anything else) there will be  
no issues. At very high concentrations the accuracy and therefore  
consistency may become lower. Concentrations between 5 and 10  
mg/ml should be fine. The instrument is pricey though.


 Vaheh



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf  
Of Filip Van Petegem

Sent: Thursday, June 16, 2011 3:34 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus  
good old Bradford.


Dear Arnon,

the Bradford method is not recommended for accurate measurements.   
The readings are strongly dependent on the amino acid composition.  
 A much better method is using the absorption at 280nm under  
denaturing conditions (6M Guanidine), and using calculated  
extinction coefficients based on the composition of mostly  
Tyrosine and Tryptophan residues (+ disulfide bonds).  This method  
is also old (Edelhoch, 1967), but very reliable.


One thing about the nanodrop: smaller volume = more evaporation.   
On the demo we've had, I was so unimpressed with the precision  
(25% variability between two consecutive measurement) that we  
didn't consider this instrument at all.  So unless you just want a  
'rough' estimate, I wouldn't

Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Edward A. Berry

Arnon Lavie wrote:
~~~

We have been considering buying a Nanodrop machine (small volume, no
dilution needed, fast, easy).
However, while testing our samples using a colleague's machine, we have
gotten readings up to 100% different to our Bradford assay (all fully
purified proteins). For example, Bradford says 6 mg/ml, Nanodrop 3
mg/ml. So while it is fun/easy to use the Nanodrop, I am not sure how
reliable are the measurements (your thoughts?).

So QUESTION 1: What are people's experience regarding the correlation
between Nanodrop and Bradford?


Bradford is an assay, Nanodrop is a spectrophotometer.
Both the A280 and Bradford methods are strongly dependent on
amino acid composition, so unless you correct A280 for that
as mentioned by Filip, either one is semiquantitative.
Occasionally you come across a protein with no tryptophan
which will have a much lower extinction coefficient.
Try making a 1 g/l solution of gelatin (collagen?)
and see what its A280 is!  I noticed recently the
protparam tool at http://ca.expasy.org/cgi-bin/protparam
estimates the extinction coefficient given a sequence.



David Briggs wrote:
~~~


I wouldn't touch Bradford with a barge-pole. I've found it to be
wildly inaccurate for certain proteins I've handled, where as the
OD280 measurements have been fine.


One wonders what does fine mean, like same as with Biuret or
Kjeldahl nitrogen, or solution made up by weight?


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Filip Van Petegem
. The instrument is pricey though.

 Vaheh



 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
 Filip Van Petegem
 Sent: Thursday, June 16, 2011 3:34 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good
 old Bradford.

 Dear Arnon,

 the Bradford method is not recommended for accurate measurements.  The
 readings are strongly dependent on the amino acid composition.  A much
 better method is using the absorption at 280nm under denaturing conditions
 (6M Guanidine), and using calculated extinction coefficients based on the
 composition of mostly Tyrosine and Tryptophan residues (+ disulfide bonds).
  This method is also old (Edelhoch, 1967), but very reliable.

 One thing about the nanodrop: smaller volume = more evaporation.  On the
 demo we've had, I was so unimpressed with the precision (25% variability
 between two consecutive measurement) that we didn't consider this 
 instrument
 at all.  So unless you just want a 'rough' estimate, I wouldn't recommend 
 it
 at all. But most respectable spectrophotometers will take cuvettes with 
 50ul
 volumes - a big step up from 1ml volumes...

 Filip Van Petegem



 On Thu, Jun 16, 2011 at 12:15 PM, Arnon Lavie la...@uic.edu wrote:
 Dear fellow crystallographers - a question about spectrophotometers for
 protein concentration determination.

 We are so last millennium - using Bradford reagent/ 1 ml cuvette for
 protein conc. determination.

 We have been considering buying a Nanodrop machine (small volume, no
 dilution needed, fast, easy).
 However, while testing our samples using a colleague's machine, we have
 gotten readings up to 100% different to our Bradford assay (all fully
 purified proteins). For example, Bradford says 6 mg/ml, Nanodrop 3 mg/ml. 
 So
 while it is fun/easy to use the Nanodrop, I am not sure how reliable are 
 the
 measurements (your thoughts?).

 So QUESTION 1: What are people's experience regarding the correlation
 between Nanodrop and Bradford?

 While researching the Nanodrop machine, I heard about the Implen
 NanoPhotmeter Pearl.
 So Question 2: Is the Pearl better/worse/same as the Nanodrop for our
 purpose?

 Thank you for helping us to advance to the next millennium, even if it
 is nearly a dozen years late.

 Arnon

 --
 ***
 Arnon Lavie, Professor
 Dept. of Biochemistry and Molecular Genetics
 University of Illinois at Chicago
 900 S. Ashland Ave.
 Molecular Biology Research Building, Room 1108 (M/C 669)
 Chicago, IL 60607
 U.S.A.
Tel:(312) 355-5029
Fax:(312) 355-4535
E-mail: la...@uic.edu
http://www.uic.edu/labs/lavie/
 ***



 --
 Filip Van Petegem, PhD
 Assistant Professor
 The University of British Columbia
 Dept. of Biochemistry and Molecular Biology
 2350 Health Sciences Mall - Rm 2.356
 Vancouver, V6T 1Z3

 phone: +1 604 827 4267
 email: filip.vanpete...@gmail.com
 http://crg.ubc.ca/VanPetegem/
 To the extent this electronic communication or any of its attachments
 contain information that is not in the public domain, such information is
 considered by MedImmune to be confidential and proprietary. This
 communication is expected to be read and/or used only by the individual(s)
 for whom it is intended. If you have received this electronic communication
 in error, please reply to the sender advising of the error in transmission
 and delete the original message and any accompanying documents from your
 system immediately, without copying, reviewing or otherwise using them for
 any purpose. Thank you for your cooperation.





 --
 Filip Van Petegem, PhD
 Assistant Professor
 The University of British Columbia
 Dept. of Biochemistry and Molecular Biology
 2350 Health Sciences Mall - Rm 2.356
 Vancouver, V6T 1Z3

 phone: +1 604 827 4267
 email: filip.vanpete...@gmail.com
 http://crg.ubc.ca/VanPetegem/






-- 
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: filip.vanpete...@gmail.com
http://crg.ubc.ca/VanPetegem/


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread David Briggs
'Fine' for me means comparable to SEC-MALLS measurements and reproducible.
I use the E calculated from the sequence using the protparam server at Expasy.


David C. Briggs PhD
Father, Structural Biologist and Sceptic

University of Manchester E-mail:
david.c.bri...@manchester.ac.uk

http://manchester.academia.edu/DavidBriggs (v.sensible)
http://xtaldave.wordpress.com/ (sensible)
http://xtaldave.posterous.com/ (less sensible)
Twitter: @xtaldave
Skype: DocDCB




On 16 June 2011 22:31, Edward A. Berry ber...@upstate.edu wrote:
 Arnon Lavie wrote:
 ~~~

 We have been considering buying a Nanodrop machine (small volume, no
 dilution needed, fast, easy).
 However, while testing our samples using a colleague's machine, we have
 gotten readings up to 100% different to our Bradford assay (all fully
 purified proteins). For example, Bradford says 6 mg/ml, Nanodrop 3
 mg/ml. So while it is fun/easy to use the Nanodrop, I am not sure how
 reliable are the measurements (your thoughts?).

 So QUESTION 1: What are people's experience regarding the correlation
 between Nanodrop and Bradford?

 Bradford is an assay, Nanodrop is a spectrophotometer.
 Both the A280 and Bradford methods are strongly dependent on
 amino acid composition, so unless you correct A280 for that
 as mentioned by Filip, either one is semiquantitative.
 Occasionally you come across a protein with no tryptophan
 which will have a much lower extinction coefficient.
 Try making a 1 g/l solution of gelatin (collagen?)
 and see what its A280 is!  I noticed recently the
 protparam tool at http://ca.expasy.org/cgi-bin/protparam
 estimates the extinction coefficient given a sequence.



 David Briggs wrote:
 ~~~

 I wouldn't touch Bradford with a barge-pole. I've found it to be
 wildly inaccurate for certain proteins I've handled, where as the
 OD280 measurements have been fine.

 One wonders what does fine mean, like same as with Biuret or
 Kjeldahl nitrogen, or solution made up by weight?





Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Jacob Keller
, Oganesyan, Vaheh wrote:

 I completely disagree with Filip’s assessment. I’ve been using nanodrop
 nearly 5 years and never had inconsistency issues. If you work at 
 reasonable
 speed (if you put a drop there then lower the lever and click measure 
 before
 you do anything else) there will be no issues. At very high concentrations
 the accuracy and therefore consistency may become lower. Concentrations
 between 5 and 10 mg/ml should be fine. The instrument is pricey though.

     Vaheh



 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
 Filip Van Petegem
 Sent: Thursday, June 16, 2011 3:34 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good
 old Bradford.

 Dear Arnon,

 the Bradford method is not recommended for accurate measurements.  The
 readings are strongly dependent on the amino acid composition.  A much
 better method is using the absorption at 280nm under denaturing conditions
 (6M Guanidine), and using calculated extinction coefficients based on the
 composition of mostly Tyrosine and Tryptophan residues (+ disulfide 
 bonds).
  This method is also old (Edelhoch, 1967), but very reliable.

 One thing about the nanodrop: smaller volume = more evaporation.  On
 the demo we've had, I was so unimpressed with the precision (25%
 variability between two consecutive measurement) that we didn't consider
 this instrument at all.  So unless you just want a 'rough' estimate, I
 wouldn't recommend it at all. But most respectable spectrophotometers will
 take cuvettes with 50ul volumes - a big step up from 1ml volumes...

 Filip Van Petegem



 On Thu, Jun 16, 2011 at 12:15 PM, Arnon Lavie la...@uic.edu wrote:
 Dear fellow crystallographers - a question about spectrophotometers for
 protein concentration determination.

 We are so last millennium - using Bradford reagent/ 1 ml cuvette for
 protein conc. determination.

 We have been considering buying a Nanodrop machine (small volume, no
 dilution needed, fast, easy).
 However, while testing our samples using a colleague's machine, we have
 gotten readings up to 100% different to our Bradford assay (all fully
 purified proteins). For example, Bradford says 6 mg/ml, Nanodrop 3 mg/ml. 
 So
 while it is fun/easy to use the Nanodrop, I am not sure how reliable are 
 the
 measurements (your thoughts?).

 So QUESTION 1: What are people's experience regarding the correlation
 between Nanodrop and Bradford?

 While researching the Nanodrop machine, I heard about the Implen
 NanoPhotmeter Pearl.
 So Question 2: Is the Pearl better/worse/same as the Nanodrop for our
 purpose?

 Thank you for helping us to advance to the next millennium, even if it
 is nearly a dozen years late.

 Arnon

 --
 ***
 Arnon Lavie, Professor
 Dept. of Biochemistry and Molecular Genetics
 University of Illinois at Chicago
 900 S. Ashland Ave.
 Molecular Biology Research Building, Room 1108 (M/C 669)
 Chicago, IL 60607
 U.S.A.
                            Tel:        (312) 355-5029
                            Fax:        (312) 355-4535
                            E-mail:     la...@uic.edu
                            http://www.uic.edu/labs/lavie/
 ***



 --
 Filip Van Petegem, PhD
 Assistant Professor
 The University of British Columbia
 Dept. of Biochemistry and Molecular Biology
 2350 Health Sciences Mall - Rm 2.356
 Vancouver, V6T 1Z3

 phone: +1 604 827 4267
 email: filip.vanpete...@gmail.com
 http://crg.ubc.ca/VanPetegem/
 To the extent this electronic communication or any of its attachments
 contain information that is not in the public domain, such information is
 considered by MedImmune to be confidential and proprietary. This
 communication is expected to be read and/or used only by the individual(s)
 for whom it is intended. If you have received this electronic 
 communication
 in error, please reply to the sender advising of the error in transmission
 and delete the original message and any accompanying documents from your
 system immediately, without copying, reviewing or otherwise using them for
 any purpose. Thank you for your cooperation.




 --
 Filip Van Petegem, PhD
 Assistant Professor
 The University of British Columbia
 Dept. of Biochemistry and Molecular Biology
 2350 Health Sciences Mall - Rm 2.356
 Vancouver, V6T 1Z3

 phone: +1 604 827 4267
 email: filip.vanpete...@gmail.com
 http://crg.ubc.ca/VanPetegem/





 --
 Filip Van Petegem, PhD
 Assistant Professor
 The University of British Columbia
 Dept. of Biochemistry and Molecular Biology
 2350 Health Sciences Mall - Rm 2.356
 Vancouver, V6T 1Z3

 phone: +1 604 827 4267
 email: filip.vanpete...@gmail.com
 http://crg.ubc.ca/VanPetegem/




-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Petr Leiman
Totally support the statements below. We have had several proteins with A280 
absorbance of 0.1 or less (at 1 mg/ml). You _have_ to use Bradford in the 
Nanodrop or whatnot to measure the concentration.

Before purchasing the Nanodrop we used a Hellma TrayCell and a normal UV/Vis 
instrument. Similar to the Nanodrop, the sample volume in TrayCell is  2-3 ul. 
Traycell works well at a fraction of the Nanodrop cost, but Nanodrop is a lot 
more convenient to use for high concentration quick measurements (especially if 
you need to measure several things in succession), so you get what you pay for. 
 

Petr

P.S. Expasy's Protparam tool has been around for ages (10-12+ years?). That 
plus the Nanodrop are two essential and synergetic tools of a protein 
chemist/crystallographer. 

On Jun 16, 2011, at 10:31 PM, Edward A. Berry wrote:

 
 Bradford is an assay, Nanodrop is a spectrophotometer.
 Both the A280 and Bradford methods are strongly dependent on
 amino acid composition, so unless you correct A280 for that
 as mentioned by Filip, either one is semiquantitative.
 Occasionally you come across a protein with no tryptophan
 which will have a much lower extinction coefficient.
 Try making a 1 g/l solution of gelatin (collagen?)
 and see what its A280 is!  I noticed recently the
 protparam tool at http://ca.expasy.org/cgi-bin/protparam
 estimates the extinction coefficient given a sequence.
 
 
 
 David Briggs wrote:
 ~~~
 
 I wouldn't touch Bradford with a barge-pole. I've found it to be
 wildly inaccurate for certain proteins I've handled, where as the
 OD280 measurements have been fine.
 
 One wonders what does fine mean, like same as with Biuret or
 Kjeldahl nitrogen, or solution made up by weight?


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Machius, Mischa Christian
With respect to the Edelhoch method and the ProtParam server, I would strongly 
recommend determining extinction coefficients experimentally and not rely on 
the ProtParam values. The reason is that the underlying extinction coefficients 
in the formula used by ProtParam and referenced there are statistical averages. 
They may or may not be valid for a given protein. I have seen differences of 
more than 20% between the theoretical and experimental extinction 
coefficients, particularly for proteins with few Trp and Tyr residues. When 
relying on relative concentrations, this inaccuracy is not detrimental, but 
when absolute concentrations are needed (CD, AUC, ITC, any binding experiment, 
etc.), such a difference would be considered huge. Determining an extinction 
coefficient experimentally takes but a few minutes.

Cheers!
MM


On Jun 16, 2011, at 6:22 PM, Petr Leiman wrote:

Totally support the statements below. We have had several proteins with A280 
absorbance of 0.1 or less (at 1 mg/ml). You _have_ to use Bradford in the 
Nanodrop or whatnot to measure the concentration.

Before purchasing the Nanodrop we used a Hellma TrayCell and a normal UV/Vis 
instrument. Similar to the Nanodrop, the sample volume in TrayCell is  2-3 ul. 
Traycell works well at a fraction of the Nanodrop cost, but Nanodrop is a lot 
more convenient to use for high concentration quick measurements (especially if 
you need to measure several things in succession), so you get what you pay for.

Petr

P.S. Expasy's Protparam tool has been around for ages (10-12+ years?). That 
plus the Nanodrop are two essential and synergetic tools of a protein 
chemist/crystallographer.

On Jun 16, 2011, at 10:31 PM, Edward A. Berry wrote:


Bradford is an assay, Nanodrop is a spectrophotometer.
Both the A280 and Bradford methods are strongly dependent on
amino acid composition, so unless you correct A280 for that
as mentioned by Filip, either one is semiquantitative.
Occasionally you come across a protein with no tryptophan
which will have a much lower extinction coefficient.
Try making a 1 g/l solution of gelatin (collagen?)
and see what its A280 is!  I noticed recently the
protparam tool at http://ca.expasy.org/cgi-bin/protparam
estimates the extinction coefficient given a sequence.



David Briggs wrote:
~~~

I wouldn't touch Bradford with a barge-pole. I've found it to be
wildly inaccurate for certain proteins I've handled, where as the
OD280 measurements have been fine.

One wonders what does fine mean, like same as with Biuret or
Kjeldahl nitrogen, or solution made up by weight?

---
Mischa Machius, PhD
Director, Center for Structural Biology
Assoc. Professor, Dept. of Pharmacology
Member, Lineberger Comprehensive Cancer Center
University of North Carolina
4079 Genetic Medicine
CB#7365
120 Mason Farm Road
Chapel Hill, NC 27599-7365, U.S.A.
tel: +1-919-843-4485
fax: +1-919-966-5640
email: mach...@unc.edumailto:mach...@med.unc.edu



Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Oganesyan, Vaheh
I think that the absolute value of protein concentration is not very important. 
Some proteins get crystallized at 1 mg/ml, others at 50. What is important is 
to be able to reproducibly estimate it from prep to prep. You probably want to 
start at some reasonable value of about 10 mg/ml. If it in reality is 12.5 I 
don't personally care.
If I publish the result and someone repeats and doesn't get crystal at exactly 
same concentration I don't care either because that person is not taking 
sensible approach.

 Vaheh




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Machius, 
Mischa Christian
Sent: Thursday, June 16, 2011 7:23 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old 
Bradford.

With respect to the Edelhoch method and the ProtParam server, I would strongly 
recommend determining extinction coefficients experimentally and not rely on 
the ProtParam values. The reason is that the underlying extinction coefficients 
in the formula used by ProtParam and referenced there are statistical averages. 
They may or may not be valid for a given protein. I have seen differences of 
more than 20% between the theoretical and experimental extinction 
coefficients, particularly for proteins with few Trp and Tyr residues. When 
relying on relative concentrations, this inaccuracy is not detrimental, but 
when absolute concentrations are needed (CD, AUC, ITC, any binding experiment, 
etc.), such a difference would be considered huge. Determining an extinction 
coefficient experimentally takes but a few minutes.

Cheers!
MM


On Jun 16, 2011, at 6:22 PM, Petr Leiman wrote:


Totally support the statements below. We have had several proteins with A280 
absorbance of 0.1 or less (at 1 mg/ml). You _have_ to use Bradford in the 
Nanodrop or whatnot to measure the concentration.

Before purchasing the Nanodrop we used a Hellma TrayCell and a normal UV/Vis 
instrument. Similar to the Nanodrop, the sample volume in TrayCell is  2-3 ul. 
Traycell works well at a fraction of the Nanodrop cost, but Nanodrop is a lot 
more convenient to use for high concentration quick measurements (especially if 
you need to measure several things in succession), so you get what you pay for.

Petr

P.S. Expasy's Protparam tool has been around for ages (10-12+ years?). That 
plus the Nanodrop are two essential and synergetic tools of a protein 
chemist/crystallographer.

On Jun 16, 2011, at 10:31 PM, Edward A. Berry wrote:


Bradford is an assay, Nanodrop is a spectrophotometer.
Both the A280 and Bradford methods are strongly dependent on
amino acid composition, so unless you correct A280 for that
as mentioned by Filip, either one is semiquantitative.
Occasionally you come across a protein with no tryptophan
which will have a much lower extinction coefficient.
Try making a 1 g/l solution of gelatin (collagen?)
and see what its A280 is!  I noticed recently the
protparam tool at http://ca.expasy.org/cgi-bin/protparam
estimates the extinction coefficient given a sequence.



David Briggs wrote:
~~~

I wouldn't touch Bradford with a barge-pole. I've found it to be
wildly inaccurate for certain proteins I've handled, where as the
OD280 measurements have been fine.

One wonders what does fine mean, like same as with Biuret or
Kjeldahl nitrogen, or solution made up by weight?

---
Mischa Machius, PhD
Director, Center for Structural Biology
Assoc. Professor, Dept. of Pharmacology
Member, Lineberger Comprehensive Cancer Center
University of North Carolina
4079 Genetic Medicine
CB#7365
120 Mason Farm Road
Chapel Hill, NC 27599-7365, U.S.A.
tel: +1-919-843-4485
fax: +1-919-966-5640
email: mach...@unc.edumailto:mach...@med.unc.edu

To the extent this electronic communication or any of its attachments contain 
information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary. This communication is expected to 
be read and/or used only by the individual(s) for whom it is intended. If you 
have received this electronic communication in error, please reply to the 
sender advising of the error in transmission and delete the original message 
and any accompanying documents from your system immediately, without copying, 
reviewing or otherwise using them for any purpose. Thank you for your 
cooperation.


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread aaleshin
Mischa, 
You intrigued me. What is the experimental technique for the Extinction 
Coefficient  measurement (which requires knowledge of protein concentration)? 
Let me guess, Bradford? Protein evaporation and weighing? 

Alex

On Jun 16, 2011, at 4:22 PM, Machius, Mischa Christian wrote:

 With respect to the Edelhoch method and the ProtParam server, I would 
 strongly recommend determining extinction coefficients experimentally and not 
 rely on the ProtParam values. The reason is that the underlying extinction 
 coefficients in the formula used by ProtParam and referenced there are 
 statistical averages. They may or may not be valid for a given protein. I 
 have seen differences of more than 20% between the theoretical and 
 experimental extinction coefficients, particularly for proteins with few 
 Trp and Tyr residues. When relying on relative concentrations, this 
 inaccuracy is not detrimental, but when absolute concentrations are needed 
 (CD, AUC, ITC, any binding experiment, etc.), such a difference would be 
 considered huge. Determining an extinction coefficient experimentally takes 
 but a few minutes.
 
 Cheers!
 MM
 
 
 On Jun 16, 2011, at 6:22 PM, Petr Leiman wrote:
 
 Totally support the statements below. We have had several proteins with A280 
 absorbance of 0.1 or less (at 1 mg/ml). You _have_ to use Bradford in the 
 Nanodrop or whatnot to measure the concentration.
 
 Before purchasing the Nanodrop we used a Hellma TrayCell and a normal 
 UV/Vis instrument. Similar to the Nanodrop, the sample volume in TrayCell is 
  2-3 ul. Traycell works well at a fraction of the Nanodrop cost, but 
 Nanodrop is a lot more convenient to use for high concentration quick 
 measurements (especially if you need to measure several things in 
 succession), so you get what you pay for.  
 
 Petr
 
 P.S. Expasy's Protparam tool has been around for ages (10-12+ years?). That 
 plus the Nanodrop are two essential and synergetic tools of a protein 
 chemist/crystallographer. 
 
 On Jun 16, 2011, at 10:31 PM, Edward A. Berry wrote:
 
 
 Bradford is an assay, Nanodrop is a spectrophotometer.
 Both the A280 and Bradford methods are strongly dependent on
 amino acid composition, so unless you correct A280 for that
 as mentioned by Filip, either one is semiquantitative.
 Occasionally you come across a protein with no tryptophan
 which will have a much lower extinction coefficient.
 Try making a 1 g/l solution of gelatin (collagen?)
 and see what its A280 is!  I noticed recently the
 protparam tool at http://ca.expasy.org/cgi-bin/protparam
 estimates the extinction coefficient given a sequence.
 
 
 
 David Briggs wrote:
 ~~~
 
 I wouldn't touch Bradford with a barge-pole. I've found it to be
 wildly inaccurate for certain proteins I've handled, where as the
 OD280 measurements have been fine.
 
 One wonders what does fine mean, like same as with Biuret or
 Kjeldahl nitrogen, or solution made up by weight?
 
 ---
 Mischa Machius, PhD
 Director, Center for Structural Biology
 Assoc. Professor, Dept. of Pharmacology
 Member, Lineberger Comprehensive Cancer Center
 University of North Carolina
 4079 Genetic Medicine
 CB#7365
 120 Mason Farm Road
 Chapel Hill, NC 27599-7365, U.S.A.
 tel: +1-919-843-4485
 fax: +1-919-966-5640
 email: mach...@unc.edu
 



Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread aaleshin
Sorry for misprint, I meant evaporating water from a protein solution...

On Jun 16, 2011, at 4:45 PM, aaleshin wrote:

 Mischa, 
 You intrigued me. What is the experimental technique for the Extinction 
 Coefficient  measurement (which requires knowledge of protein concentration)? 
 Let me guess, Bradford? Protein evaporation and weighing? 
 
 Alex
 
 On Jun 16, 2011, at 4:22 PM, Machius, Mischa Christian wrote:
 
 With respect to the Edelhoch method and the ProtParam server, I would 
 strongly recommend determining extinction coefficients experimentally and 
 not rely on the ProtParam values. The reason is that the underlying 
 extinction coefficients in the formula used by ProtParam and referenced 
 there are statistical averages. They may or may not be valid for a given 
 protein. I have seen differences of more than 20% between the theoretical 
 and experimental extinction coefficients, particularly for proteins with 
 few Trp and Tyr residues. When relying on relative concentrations, this 
 inaccuracy is not detrimental, but when absolute concentrations are needed 
 (CD, AUC, ITC, any binding experiment, etc.), such a difference would be 
 considered huge. Determining an extinction coefficient experimentally takes 
 but a few minutes.
 
 Cheers!
 MM
 
 
 On Jun 16, 2011, at 6:22 PM, Petr Leiman wrote:
 
 Totally support the statements below. We have had several proteins with 
 A280 absorbance of 0.1 or less (at 1 mg/ml). You _have_ to use Bradford in 
 the Nanodrop or whatnot to measure the concentration.
 
 Before purchasing the Nanodrop we used a Hellma TrayCell and a normal 
 UV/Vis instrument. Similar to the Nanodrop, the sample volume in TrayCell 
 is  2-3 ul. Traycell works well at a fraction of the Nanodrop cost, but 
 Nanodrop is a lot more convenient to use for high concentration quick 
 measurements (especially if you need to measure several things in 
 succession), so you get what you pay for.  
 
 Petr
 
 P.S. Expasy's Protparam tool has been around for ages (10-12+ years?). That 
 plus the Nanodrop are two essential and synergetic tools of a protein 
 chemist/crystallographer. 
 
 On Jun 16, 2011, at 10:31 PM, Edward A. Berry wrote:
 
 
 Bradford is an assay, Nanodrop is a spectrophotometer.
 Both the A280 and Bradford methods are strongly dependent on
 amino acid composition, so unless you correct A280 for that
 as mentioned by Filip, either one is semiquantitative.
 Occasionally you come across a protein with no tryptophan
 which will have a much lower extinction coefficient.
 Try making a 1 g/l solution of gelatin (collagen?)
 and see what its A280 is!  I noticed recently the
 protparam tool at http://ca.expasy.org/cgi-bin/protparam
 estimates the extinction coefficient given a sequence.
 
 
 
 David Briggs wrote:
 ~~~
 
 I wouldn't touch Bradford with a barge-pole. I've found it to be
 wildly inaccurate for certain proteins I've handled, where as the
 OD280 measurements have been fine.
 
 One wonders what does fine mean, like same as with Biuret or
 Kjeldahl nitrogen, or solution made up by weight?
 
 ---
 Mischa Machius, PhD
 Director, Center for Structural Biology
 Assoc. Professor, Dept. of Pharmacology
 Member, Lineberger Comprehensive Cancer Center
 University of North Carolina
 4079 Genetic Medicine
 CB#7365
 120 Mason Farm Road
 Chapel Hill, NC 27599-7365, U.S.A.
 tel: +1-919-843-4485
 fax: +1-919-966-5640
 email: mach...@unc.edu
 
 



Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Filip Van Petegem
A convenient fast way is the earlier mentioned Edelhoch method, as described
in this paper which is referenced on the popular Protparam tool:

http://onlinelibrary.wiley.com/doi/10.1002/pro.5560041120/pdf


Filip

On Thu, Jun 16, 2011 at 4:45 PM, aaleshin aales...@burnham.org wrote:

 Mischa,
 You intrigued me. What is the experimental technique for the Extinction
 Coefficient  measurement (which requires knowledge of protein
 concentration)? Let me guess, Bradford? Protein evaporation and weighing?

 Alex


 On Jun 16, 2011, at 4:22 PM, Machius, Mischa Christian wrote:

 With respect to the Edelhoch method and the ProtParam server, I would
 strongly recommend determining extinction coefficients experimentally and
 not rely on the ProtParam values. The reason is that the underlying
 extinction coefficients in the formula used by ProtParam and referenced
 there are statistical averages. They may or may not be valid for a given
 protein. I have seen differences of more than 20% between the theoretical
 and experimental extinction coefficients, particularly for proteins with
 few Trp and Tyr residues. When relying on relative concentrations, this
 inaccuracy is not detrimental, but when absolute concentrations are needed
 (CD, AUC, ITC, any binding experiment, etc.), such a difference would be
 considered huge. Determining an extinction coefficient experimentally takes
 but a few minutes.

 Cheers!
 MM


 On Jun 16, 2011, at 6:22 PM, Petr Leiman wrote:

 Totally support the statements below. We have had several proteins with
 A280 absorbance of 0.1 or less (at 1 mg/ml). You _have_ to use Bradford in
 the Nanodrop or whatnot to measure the concentration.


  Before purchasing the Nanodrop we used a Hellma TrayCell and a normal
 UV/Vis instrument. Similar to the Nanodrop, the sample volume in TrayCell is
  2-3 ul. Traycell works well at a fraction of the Nanodrop cost, but
 Nanodrop is a lot more convenient to use for high concentration quick
 measurements (especially if you need to measure several things in
 succession), so you get what you pay for.


  Petr


  P.S. Expasy's Protparam tool has been around for ages (10-12+ years?).
 That plus the Nanodrop are two essential and synergetic tools of a protein
 chemist/crystallographer.


  On Jun 16, 2011, at 10:31 PM, Edward A. Berry wrote:



   Bradford is an assay, Nanodrop is a spectrophotometer.

  Both the A280 and Bradford methods are strongly dependent on

  amino acid composition, so unless you correct A280 for that

  as mentioned by Filip, either one is semiquantitative.

  Occasionally you come across a protein with no tryptophan

  which will have a much lower extinction coefficient.

  Try making a 1 g/l solution of gelatin (collagen?)

  and see what its A280 is!  I noticed recently the

  protparam tool at http://ca.expasy.org/cgi-bin/protparam

  estimates the extinction coefficient given a sequence.




   David Briggs wrote:

  ~~~


I wouldn't touch Bradford with a barge-pole. I've found it to be

   wildly inaccurate for certain proteins I've handled, where as the

   OD280 measurements have been fine.


   One wonders what does fine mean, like same as with Biuret or

  Kjeldahl nitrogen, or solution made up by weight?


  ---
  Mischa Machius, PhD
  Director, Center for Structural Biology
  Assoc. Professor, Dept. of Pharmacology
  Member, Lineberger Comprehensive Cancer Center
  University of North Carolina
  4079 Genetic Medicine
  CB#7365
  120 Mason Farm Road
  Chapel Hill, NC 27599-7365, U.S.A.
  tel: +1-919-843-4485
  fax: +1-919-966-5640
  email: mach...@unc.edu mach...@med.unc.edu





-- 
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: filip.vanpete...@gmail.com
http://crg.ubc.ca/VanPetegem/


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Shaun Lott
Just to add my 2c worth...

The department here has a couple of nanodrops as a shared facility, one for 
DNA/RNA and one for protein. It has been noticeable that over time people has 
been getting decreased reliability of measurements on the latter machine cf 
cuvette measurements, presumably due to the build-up of protein deposits over 
time - so I would say that although it's easier to clean than a cuvette, the 
nanodrop is not immune to the problem. The biggest issue I see with the 
nanodrop is evaporation of sample. Even here in moist Auckland, where RH is 
very often 80%+, taking a series of measurements with the nanodrop over a 
period of just a minute or two shows increasing concentration in the sample. 
So, for consistent results, one has to be careful to measure quickly. It's 
probably fine for comparative measurements, but as has been observed above, not 
great for super-accurate values for biophysics, and I think rather operator 
dependent. But all our students are super-careful, right? ;) Worth to note also 
that ProtParam calculates extinction coefficients based on Gill  von Hippel, 
(Gill, S.C. and von Hippel, P.H. (1989) Calculation of protein extinction 
coefficients from amino acid sequence data. Anal. Biochem. 182:319-326) who 
claim accuracy of ~5% for 'normal globular' proteins without extra 
chromophores. Whilst on this subject, I would put in a plug for the good old 
BCA (aka Pierce) assay for protein concentration. It's a little slow, but gets 
away from sequence dependency somewhat as it is primarily dependent on the 
peptide backbone rather than sidechains  and works well in micro-titre plates 
etc. It is certainly very superior to Bradford. (Smith, P.K., et al. (1985). 
Measurement of protein using bicinchoninic acid. Anal. Biochem. 150 (1): 
76–85. doi:10.1016/0003-2697(85)90442-7). 

cheers

Shaun


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Scott Pegan
Here is also a very effective method:

1Gill, S.  Hippel, P. v. Calculation of protein extinction coefficients
from amino acid sequence data. Analytical Biochemistry 182, 319-326,
(1989).


On Thu, Jun 16, 2011 at 5:56 PM, Filip Van Petegem 
filip.vanpete...@gmail.com wrote:

 A convenient fast way is the earlier mentioned Edelhoch method, as
 described in this paper which is referenced on the popular Protparam tool:

 http://onlinelibrary.wiley.com/doi/10.1002/pro.5560041120/pdf


 Filip

 On Thu, Jun 16, 2011 at 4:45 PM, aaleshin aales...@burnham.org wrote:

 Mischa,
 You intrigued me. What is the experimental technique for the Extinction
 Coefficient  measurement (which requires knowledge of protein
 concentration)? Let me guess, Bradford? Protein evaporation and weighing?

 Alex


 On Jun 16, 2011, at 4:22 PM, Machius, Mischa Christian wrote:

 With respect to the Edelhoch method and the ProtParam server, I would
 strongly recommend determining extinction coefficients experimentally and
 not rely on the ProtParam values. The reason is that the underlying
 extinction coefficients in the formula used by ProtParam and referenced
 there are statistical averages. They may or may not be valid for a given
 protein. I have seen differences of more than 20% between the theoretical
 and experimental extinction coefficients, particularly for proteins with
 few Trp and Tyr residues. When relying on relative concentrations, this
 inaccuracy is not detrimental, but when absolute concentrations are needed
 (CD, AUC, ITC, any binding experiment, etc.), such a difference would be
 considered huge. Determining an extinction coefficient experimentally takes
 but a few minutes.

 Cheers!
 MM


 On Jun 16, 2011, at 6:22 PM, Petr Leiman wrote:

 Totally support the statements below. We have had several proteins with
 A280 absorbance of 0.1 or less (at 1 mg/ml). You _have_ to use Bradford in
 the Nanodrop or whatnot to measure the concentration.


  Before purchasing the Nanodrop we used a Hellma TrayCell and a normal
 UV/Vis instrument. Similar to the Nanodrop, the sample volume in TrayCell is
  2-3 ul. Traycell works well at a fraction of the Nanodrop cost, but
 Nanodrop is a lot more convenient to use for high concentration quick
 measurements (especially if you need to measure several things in
 succession), so you get what you pay for.


  Petr


  P.S. Expasy's Protparam tool has been around for ages (10-12+ years?).
 That plus the Nanodrop are two essential and synergetic tools of a protein
 chemist/crystallographer.


  On Jun 16, 2011, at 10:31 PM, Edward A. Berry wrote:



   Bradford is an assay, Nanodrop is a spectrophotometer.

  Both the A280 and Bradford methods are strongly dependent on

  amino acid composition, so unless you correct A280 for that

  as mentioned by Filip, either one is semiquantitative.

  Occasionally you come across a protein with no tryptophan

  which will have a much lower extinction coefficient.

  Try making a 1 g/l solution of gelatin (collagen?)

  and see what its A280 is!  I noticed recently the

  protparam tool at http://ca.expasy.org/cgi-bin/protparam

  estimates the extinction coefficient given a sequence.




   David Briggs wrote:

  ~~~


I wouldn't touch Bradford with a barge-pole. I've found it to be

   wildly inaccurate for certain proteins I've handled, where as the

   OD280 measurements have been fine.


   One wonders what does fine mean, like same as with Biuret or

  Kjeldahl nitrogen, or solution made up by weight?


  ---
  Mischa Machius, PhD
  Director, Center for Structural Biology
  Assoc. Professor, Dept. of Pharmacology
  Member, Lineberger Comprehensive Cancer Center
  University of North Carolina
  4079 Genetic Medicine
  CB#7365
  120 Mason Farm Road
  Chapel Hill, NC 27599-7365, U.S.A.
  tel: +1-919-843-4485
  fax: +1-919-966-5640
  email: mach...@unc.edu mach...@med.unc.edu





 --
 Filip Van Petegem, PhD
 Assistant Professor
 The University of British Columbia
 Dept. of Biochemistry and Molecular Biology
 2350 Health Sciences Mall - Rm 2.356
 Vancouver, V6T 1Z3

 phone: +1 604 827 4267
 email: filip.vanpete...@gmail.com
 http://crg.ubc.ca/VanPetegem/




-- 
Scott D. Pegan, Ph.D.
Assistant Professor
Chemistry  Biochemistry
University of Denver
Office: 303 871 2533
Fax: 303 871 2254


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Machius, Mischa Christian
The method is that by Edelhoch, mentioned a couple of times already in this 
discussion. It's also described in the paper by Pace et al., the same paper 
that the formula in ProtParam is from (ProtParam does not use the values 
determined by Gill  von Hippel). Last time I looked into this, the consensus 
was that the Edelhoch method is the most accurate method for protein 
concentration determination; more accurate than dry-weighing plus N-terminal 
sequencing, etc.

MM


On Jun 16, 2011, at 7:51 PM, aaleshin wrote:

Sorry for misprint, I meant evaporating water from a protein solution...

On Jun 16, 2011, at 4:45 PM, aaleshin wrote:

Mischa,
You intrigued me. What is the experimental technique for the Extinction 
Coefficient  measurement (which requires knowledge of protein concentration)? 
Let me guess, Bradford? Protein evaporation and weighing?

Alex

On Jun 16, 2011, at 4:22 PM, Machius, Mischa Christian wrote:

With respect to the Edelhoch method and the ProtParam server, I would strongly 
recommend determining extinction coefficients experimentally and not rely on 
the ProtParam values. The reason is that the underlying extinction coefficients 
in the formula used by ProtParam and referenced there are statistical averages. 
They may or may not be valid for a given protein. I have seen differences of 
more than 20% between the theoretical and experimental extinction 
coefficients, particularly for proteins with few Trp and Tyr residues. When 
relying on relative concentrations, this inaccuracy is not detrimental, but 
when absolute concentrations are needed (CD, AUC, ITC, any binding experiment, 
etc.), such a difference would be considered huge. Determining an extinction 
coefficient experimentally takes but a few minutes.

Cheers!
MM


On Jun 16, 2011, at 6:22 PM, Petr Leiman wrote:

Totally support the statements below. We have had several proteins with A280 
absorbance of 0.1 or less (at 1 mg/ml). You _have_ to use Bradford in the 
Nanodrop or whatnot to measure the concentration.

Before purchasing the Nanodrop we used a Hellma TrayCell and a normal UV/Vis 
instrument. Similar to the Nanodrop, the sample volume in TrayCell is  2-3 ul. 
Traycell works well at a fraction of the Nanodrop cost, but Nanodrop is a lot 
more convenient to use for high concentration quick measurements (especially if 
you need to measure several things in succession), so you get what you pay for.

Petr

P.S. Expasy's Protparam tool has been around for ages (10-12+ years?). That 
plus the Nanodrop are two essential and synergetic tools of a protein 
chemist/crystallographer.

On Jun 16, 2011, at 10:31 PM, Edward A. Berry wrote:


Bradford is an assay, Nanodrop is a spectrophotometer.
Both the A280 and Bradford methods are strongly dependent on
amino acid composition, so unless you correct A280 for that
as mentioned by Filip, either one is semiquantitative.
Occasionally you come across a protein with no tryptophan
which will have a much lower extinction coefficient.
Try making a 1 g/l solution of gelatin (collagen?)
and see what its A280 is!  I noticed recently the
protparam tool at http://ca.expasy.org/cgi-bin/protparam
estimates the extinction coefficient given a sequence.



David Briggs wrote:
~~~

I wouldn't touch Bradford with a barge-pole. I've found it to be
wildly inaccurate for certain proteins I've handled, where as the
OD280 measurements have been fine.

One wonders what does fine mean, like same as with Biuret or
Kjeldahl nitrogen, or solution made up by weight?

---
Mischa Machius, PhD
Director, Center for Structural Biology
Assoc. Professor, Dept. of Pharmacology
Member, Lineberger Comprehensive Cancer Center
University of North Carolina
4079 Genetic Medicine
CB#7365
120 Mason Farm Road
Chapel Hill, NC 27599-7365, U.S.A.
tel: +1-919-843-4485
fax: +1-919-966-5640
email: mach...@unc.edumailto:mach...@med.unc.edu




---
Mischa Machius, PhD
Director, Center for Structural Biology
Assoc. Professor, Dept. of Pharmacology
Member, Lineberger Comprehensive Cancer Center
University of North Carolina
4079 Genetic Medicine
CB#7365
120 Mason Farm Road
Chapel Hill, NC 27599-7365, U.S.A.
tel: +1-919-843-4485
fax: +1-919-966-5640
email: mach...@unc.edumailto:mach...@med.unc.edu



Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Pascal Egea
I would like to add something about the NanoDrop versus NanoPearl,

I don't think that the path length is fixed on this instrument (the
NanoDrop) since if I recall well, the instruments sets the path length as it
scans through the droplet, hence the characteristic clicky noise that you
hear as the handle moves. This instrument requires recalibration every year
(or so according to the vendors, and this is of course not cheap) since
there is a moving part that can get out of alignment.
On the other hand, the NanoPear from Implen as a fixed geometry for the same
tiny amount of sample required. Thus you do not have to deal with moving
parts and recalibration.
We just bought a NanoPearl and I should also mention that this instrument
does both things: nano-drop size measurement like the NanoVue AND cuvette
size measurements (for OD600 or old school Bradford).

All the best,

On Thu, Jun 16, 2011 at 4:57 PM, Shaun Lott s.l...@auckland.ac.nz wrote:

 Just to add my 2c worth...

 The department here has a couple of nanodrops as a shared facility, one for
 DNA/RNA and one for protein. It has been noticeable that over time people
 has been getting decreased reliability of measurements on the latter machine
 cf cuvette measurements, presumably due to the build-up of protein deposits
 over time - so I would say that although it's easier to clean than a
 cuvette, the nanodrop is not immune to the problem. The biggest issue I see
 with the nanodrop is evaporation of sample. Even here in moist Auckland,
 where RH is very often 80%+, taking a series of measurements with the
 nanodrop over a period of just a minute or two shows increasing
 concentration in the sample. So, for consistent results, one has to be
 careful to measure quickly. It's probably fine for comparative measurements,
 but as has been observed above, not great for super-accurate values for
 biophysics, and I think rather operator dependent. But all our students are
 super-careful, right? ;) Worth to note also that ProtParam calculates
 extinction coefficients based on Gill  von Hippel, (Gill, S.C. and von
 Hippel, P.H. (1989) Calculation of protein extinction coefficients from
 amino acid sequence data. Anal. Biochem. 182:319-326) who claim accuracy of
 ~5% for 'normal globular' proteins without extra chromophores. Whilst on
 this subject, I would put in a plug for the good old BCA (aka Pierce) assay
 for protein concentration. It's a little slow, but gets away from sequence
 dependency somewhat as it is primarily dependent on the peptide backbone
 rather than sidechains  and works well in micro-titre plates etc. It is
 certainly very superior to Bradford. (Smith, P.K., et al. (1985).
 Measurement of protein using bicinchoninic acid. Anal. Biochem. 150 (1):
 76–85. doi:10.1016/0003-2697(85)90442-7).

 cheers

 Shaun




-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Machius, Mischa Christian
Again, the method described by Gill  von Hippel is based on statistical 
averages. Mach et al. (Anal. Biochem. 1992, 200, 74) later revised these 
values. Pace et al. (Protein Science, 1995, 4, 2411) again re-determined these 
averages, so if anything, the values from Pace should be used. Pace also refers 
back to the Edelhoch method as the most reliable method.

Here is the abstract from Pace's paper:

The molar absorption coefficient, epsilon, of a protein is usually based on 
concentrations measured by dry weight, nitrogen, or amino acid analysis. The 
studies reported here suggest that the Edelhoch method is the best method for 
measuring epsilon for a protein. (This method is described by Gill and von 
Hippel [1989, Anal Biochem 182:319- 3261 and is based on data from Edelhoch 
[1967, Biochemistry 6:1948-19541.) The absorbance of a protein at 280 nm 
depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average 
epsilon values for these chromophores in a sample of 18 well-characterized 
proteins have been estimated, and the epsilon values in water, propanol, 6 M 
guanidine hydrochloride (GdnHCI), and 8 M urea have been measured. For Trp, the 
average epsilon  values for the proteins are less than the epsilon values 
measured in any of the solvents. For Tyr, the average epsilon values for the 
proteins are intermediate between those measured in 6 M GdnHCl and those 
measured in propanol. Based on a sample of 116 measured epsilon values for 80 
proteins, the epsilon at 280 nm of a folded protein in water, epsilon(280), can 
best be predicted with this equation:

epsilon (280) M-1 cm-1 = (#Trp)(5,500) + (#Tyr)(1,490) + (#cystine)(125)

These epsilon(280) values are quite reliable for proteins containing Trp 
residues, and less reliable for proteins that do not. However, the Edelhoch 
method is convenient and accurate, and the best approach is to measure rather 
than predict epsilon.


Cheers!
MM


On Jun 16, 2011, at 8:05 PM, Scott Pegan wrote:

Here is also a very effective method:

1Gill, S.  Hippel, P. v. Calculation of protein extinction coefficients 
from amino acid sequence data. Analytical Biochemistry 182, 319-326, (1989).


On Thu, Jun 16, 2011 at 5:56 PM, Filip Van Petegem 
filip.vanpete...@gmail.commailto:filip.vanpete...@gmail.com wrote:
A convenient fast way is the earlier mentioned Edelhoch method, as described in 
this paper which is referenced on the popular Protparam tool:

http://onlinelibrary.wiley.com/doi/10.1002/pro.5560041120/pdf


Filip

On Thu, Jun 16, 2011 at 4:45 PM, aaleshin 
aales...@burnham.orgmailto:aales...@burnham.org wrote:
Mischa,
You intrigued me. What is the experimental technique for the Extinction 
Coefficient  measurement (which requires knowledge of protein concentration)? 
Let me guess, Bradford? Protein evaporation and weighing?

Alex


On Jun 16, 2011, at 4:22 PM, Machius, Mischa Christian wrote:

With respect to the Edelhoch method and the ProtParam server, I would strongly 
recommend determining extinction coefficients experimentally and not rely on 
the ProtParam values. The reason is that the underlying extinction coefficients 
in the formula used by ProtParam and referenced there are statistical averages. 
They may or may not be valid for a given protein. I have seen differences of 
more than 20% between the theoretical and experimental extinction 
coefficients, particularly for proteins with few Trp and Tyr residues. When 
relying on relative concentrations, this inaccuracy is not detrimental, but 
when absolute concentrations are needed (CD, AUC, ITC, any binding experiment, 
etc.), such a difference would be considered huge. Determining an extinction 
coefficient experimentally takes but a few minutes.

Cheers!
MM


On Jun 16, 2011, at 6:22 PM, Petr Leiman wrote:

Totally support the statements below. We have had several proteins with A280 
absorbance of 0.1 or less (at 1 mg/ml). You _have_ to use Bradford in the 
Nanodrop or whatnot to measure the concentration.

Before purchasing the Nanodrop we used a Hellma TrayCell and a normal UV/Vis 
instrument. Similar to the Nanodrop, the sample volume in TrayCell is  2-3 ul. 
Traycell works well at a fraction of the Nanodrop cost, but Nanodrop is a lot 
more convenient to use for high concentration quick measurements (especially if 
you need to measure several things in succession), so you get what you pay for.

Petr

P.S. Expasy's Protparam tool has been around for ages (10-12+ years?). That 
plus the Nanodrop are two essential and synergetic tools of a protein 
chemist/crystallographer.

On Jun 16, 2011, at 10:31 PM, Edward A. Berry wrote:


Bradford is an assay, Nanodrop is a spectrophotometer.
Both the A280 and Bradford methods are strongly dependent on
amino acid composition, so unless you correct A280 for that
as mentioned by Filip, either one is semiquantitative.
Occasionally you come across a protein with no tryptophan
which will have a much lower extinction coefficient.
Try making 

Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread aaleshin
I see, by the experimental determination of the extinction coefficient you mean 
correction for the difference between unfolded (which can be computed 
accurately) and folded proteins. Am I right?

Sorry for making this topic viral...
Alex

On Jun 16, 2011, at 5:06 PM, Machius, Mischa Christian wrote:

 The method is that by Edelhoch, mentioned a couple of times already in this 
 discussion. It's also described in the paper by Pace et al., the same paper 
 that the formula in ProtParam is from (ProtParam does not use the values 
 determined by Gill  von Hippel). Last time I looked into this, the consensus 
 was that the Edelhoch method is the most accurate method for protein 
 concentration determination; more accurate than dry-weighing plus N-terminal 
 sequencing, etc.
 
 MM
 
 
 On Jun 16, 2011, at 7:51 PM, aaleshin wrote:
 
 Sorry for misprint, I meant evaporating water from a protein solution...
 
 On Jun 16, 2011, at 4:45 PM, aaleshin wrote:
 
 Mischa, 
 You intrigued me. What is the experimental technique for the Extinction 
 Coefficient  measurement (which requires knowledge of protein 
 concentration)? Let me guess, Bradford? Protein evaporation and weighing? 
 
 Alex
 
 On Jun 16, 2011, at 4:22 PM, Machius, Mischa Christian wrote:
 
 With respect to the Edelhoch method and the ProtParam server, I would 
 strongly recommend determining extinction coefficients experimentally and 
 not rely on the ProtParam values. The reason is that the underlying 
 extinction coefficients in the formula used by ProtParam and referenced 
 there are statistical averages. They may or may not be valid for a given 
 protein. I have seen differences of more than 20% between the 
 theoretical and experimental extinction coefficients, particularly for 
 proteins with few Trp and Tyr residues. When relying on relative 
 concentrations, this inaccuracy is not detrimental, but when absolute 
 concentrations are needed (CD, AUC, ITC, any binding experiment, etc.), 
 such a difference would be considered huge. Determining an extinction 
 coefficient experimentally takes but a few minutes.
 
 Cheers!
 MM
 
 
 On Jun 16, 2011, at 6:22 PM, Petr Leiman wrote:
 
 Totally support the statements below. We have had several proteins with 
 A280 absorbance of 0.1 or less (at 1 mg/ml). You _have_ to use Bradford 
 in the Nanodrop or whatnot to measure the concentration.
 
 Before purchasing the Nanodrop we used a Hellma TrayCell and a normal 
 UV/Vis instrument. Similar to the Nanodrop, the sample volume in TrayCell 
 is  2-3 ul. Traycell works well at a fraction of the Nanodrop cost, but 
 Nanodrop is a lot more convenient to use for high concentration quick 
 measurements (especially if you need to measure several things in 
 succession), so you get what you pay for.  
 
 Petr
 
 P.S. Expasy's Protparam tool has been around for ages (10-12+ years?). 
 That plus the Nanodrop are two essential and synergetic tools of a 
 protein chemist/crystallographer. 
 
 On Jun 16, 2011, at 10:31 PM, Edward A. Berry wrote:
 
 
 Bradford is an assay, Nanodrop is a spectrophotometer.
 Both the A280 and Bradford methods are strongly dependent on
 amino acid composition, so unless you correct A280 for that
 as mentioned by Filip, either one is semiquantitative.
 Occasionally you come across a protein with no tryptophan
 which will have a much lower extinction coefficient.
 Try making a 1 g/l solution of gelatin (collagen?)
 and see what its A280 is!  I noticed recently the
 protparam tool at http://ca.expasy.org/cgi-bin/protparam
 estimates the extinction coefficient given a sequence.
 
 
 
 David Briggs wrote:
 ~~~
 
 I wouldn't touch Bradford with a barge-pole. I've found it to be
 wildly inaccurate for certain proteins I've handled, where as the
 OD280 measurements have been fine.
 
 One wonders what does fine mean, like same as with Biuret or
 Kjeldahl nitrogen, or solution made up by weight?
 
 ---
 Mischa Machius, PhD
 Director, Center for Structural Biology
 Assoc. Professor, Dept. of Pharmacology
 Member, Lineberger Comprehensive Cancer Center
 University of North Carolina
 4079 Genetic Medicine
 CB#7365
 120 Mason Farm Road
 Chapel Hill, NC 27599-7365, U.S.A.
 tel: +1-919-843-4485
 fax: +1-919-966-5640
 email: mach...@unc.edu
 
 
 
 
 ---
 Mischa Machius, PhD
 Director, Center for Structural Biology
 Assoc. Professor, Dept. of Pharmacology
 Member, Lineberger Comprehensive Cancer Center
 University of North Carolina
 4079 Genetic Medicine
 CB#7365
 120 Mason Farm Road
 Chapel Hill, NC 27599-7365, U.S.A.
 tel: +1-919-843-4485
 fax: +1-919-966-5640
 email: mach...@unc.edu
 



Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Dima Klenchin
The method is that by Edelhoch, mentioned a couple of times already in 
this discussion.


You recommended determining extinction coefficients experimentally. How 
is plugging number of specific residues into a formula constitute 
experimental determination?


It's also described in the paper by Pace et al., the same paper that the 
formula in ProtParam is from (ProtParam does not use the values determined 
by Gill  von Hippel). Last time I looked into this, the consensus was 
that the Edelhoch method is the most accurate method for protein 
concentration determination; more accurate than dry-weighing plus 
N-terminal sequencing, etc.


Nothing beats quantitative amino acid determination but it's one technique 
that requires specialized lab and good standartization. By now, it's almost 
a lost art.


I agree with Vaheh that absolute protein concentration is not critical in 
crystallization but let's not forget that the protein concentration enters, 
one way or another, into measurement of virtually every biochemical 
constant. 50-100% errors there can be extremely important.


- Dima


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Machius, Mischa Christian

On Jun 16, 2011, at 8:30 PM, Dima Klenchin wrote:

You recommended determining extinction coefficients experimentally. How is 
plugging number of specific residues into a formula constitute experimental 
determination?


That is a deeply philosophical question!

Eventually, you'll be plugging in numbers into a formula in any method. At 
least, the Edelhoch method requires you to do an experiment, whereas Gill  von 
Hippel and Pace et al. don't. Ultimately, though, all these methods are based 
on experiments that someone somewhere executed.


Nothing beats quantitative amino acid determination but it's one technique that 
requires specialized lab and good standartization. By now, it's almost a lost 
art.


The distinction that I am trying to make, and that I am apparently having 
trouble conveying, is that the theoretical methods presented by Gill  von 
Hippel and Pace et al. are based on statistical averages and thus may or may 
not be valid for a given protein, whereas the Edelhoch method is specific to a 
given protein and has been shown by Pace at al. to be the most accurate method 
(more accurate than quantitative amino acid determination, even when using a 
specialized lab and good standardization.

Three times the charm.


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Chelsy Prince
Hi everyone,

 

I am working with a membrane protein and normally measure my protein
concentration by diluting and then reading OD280 (1cm pathlength). I have
found this to be very consistent, if a bit time consuming.

 

At the syncatron, I had to use a Nanodrop for the first time to check some
SAXS dilutions I made on site and it was all over the map. We tested each
sample 8-10 times and saw large variations (i.e. 2.5-4.1mg/mL) on a single
sample. I used the Nanodrop to measure OD280 and calibrated it with the same
extinction coefficient I normally use. 

 

Since I have dodecyl-maltoside in all of my solutions (which is the cause of
most of my problems), I was wondering if it was also the culprit here. 

The detergent is probably lowering the surface tension of the drops.

 

It is possible that I was just in-experienced, but I had multiple people
from the facility helping me and we all had the same issue with my samples.
In addition, the Nanodrop would regularly complain about inconsistency
between the two readings and wouldn't accept readings/blanks at all. 

 

Is it me or the membrane protein? And is there anything that I could do to
improve the readings the next time that I need to use it?

 

Thanks,

Chelsy

 

 

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pascal
Egea
Sent: June-16-11 8:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old
Bradford.

 

I would like to add something about the NanoDrop versus NanoPearl,

 

I don't think that the path length is fixed on this instrument (the
NanoDrop) since if I recall well, the instruments sets the path length as it
scans through the droplet, hence the characteristic clicky noise that you
hear as the handle moves. This instrument requires recalibration every year
(or so according to the vendors, and this is of course not cheap) since
there is a moving part that can get out of alignment.

On the other hand, the NanoPear from Implen as a fixed geometry for the same
tiny amount of sample required. Thus you do not have to deal with moving
parts and recalibration.

We just bought a NanoPearl and I should also mention that this instrument
does both things: nano-drop size measurement like the NanoVue AND cuvette
size measurements (for OD600 or old school Bradford).

 

All the best,

 

On Thu, Jun 16, 2011 at 4:57 PM, Shaun Lott s.l...@auckland.ac.nz wrote:

Just to add my 2c worth...

The department here has a couple of nanodrops as a shared facility, one for
DNA/RNA and one for protein. It has been noticeable that over time people
has been getting decreased reliability of measurements on the latter machine
cf cuvette measurements, presumably due to the build-up of protein deposits
over time - so I would say that although it's easier to clean than a
cuvette, the nanodrop is not immune to the problem. The biggest issue I see
with the nanodrop is evaporation of sample. Even here in moist Auckland,
where RH is very often 80%+, taking a series of measurements with the
nanodrop over a period of just a minute or two shows increasing
concentration in the sample. So, for consistent results, one has to be
careful to measure quickly. It's probably fine for comparative measurements,
but as has been observed above, not great for super-accurate values for
biophysics, and I think rather operator dependent. But all our students are
super-careful, right? ;) Worth to note also that ProtParam calculates
extinction coefficients based on Gill  von Hippel, (Gill, S.C. and von
Hippel, P.H. (1989) Calculation of protein extinction coefficients from
amino acid sequence data. Anal. Biochem. 182:319-326) who claim accuracy of
~5% for 'normal globular' proteins without extra chromophores. Whilst on
this subject, I would put in a plug for the good old BCA (aka Pierce) assay
for protein concentration. It's a little slow, but gets away from sequence
dependency somewhat as it is primarily dependent on the peptide backbone
rather than sidechains  and works well in micro-titre plates etc. It is
certainly very superior to Bradford. (Smith, P.K., et al. (1985).
Measurement of protein using bicinchoninic acid. Anal. Biochem. 150 (1):
76-85. doi:10.1016/0003-2697(85)90442-7).

cheers

Shaun




-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building 
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu



Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Richard Edward Gillilan

Hi Chelsy, yes we had a lot of trouble with the nanoview during that run.  Even 
after going through the calibration procedure with the special fluid provided, 
we still had inconsistent results even on standards.  Finally, I carefully 
cleaned the return light path of the instrument (a separate set of holes 
towards the back of the glass plates). This seemed to fix the problem. The 
manual warns that users should always wipe from back to front when cleaning out 
sample. The instrument also seems sensitive to how well the drop is formed on 
the glass plate. Once the hydrophobic coat is worn or dirty, the drops spread 
and give poor results, so maybe detergent was also an issue.  So these machines 
can be fussy we are learning.

Richard Gillilan
MacCHESS


On Jun 16, 2011, at 9:03 PM, Chelsy Prince wrote:

Hi everyone,

I am working with a membrane protein and normally measure my protein 
concentration by diluting and then reading OD280 (1cm pathlength). I have found 
this to be very consistent, if a bit time consuming.

At the syncatron, I had to use a Nanodrop for the first time to check some SAXS 
dilutions I made on site and it was all over the map. We tested each sample 
8-10 times and saw large variations (i.e. 2.5-4.1mg/mL) on a single sample. I 
used the Nanodrop to measure OD280 and calibrated it with the same extinction 
coefficient I normally use.

Since I have dodecyl-maltoside in all of my solutions (which is the cause of 
most of my problems), I was wondering if it was also the culprit here.
The detergent is probably lowering the surface tension of the drops.

It is possible that I was just in-experienced, but I had multiple people from 
the facility helping me and we all had the same issue with my samples. In 
addition, the Nanodrop would regularly complain about inconsistency between the 
two readings and wouldn’t accept readings/blanks at all.

Is it me or the membrane protein? And is there anything that I could do to 
improve the readings the next time that I need to use it?

Thanks,
Chelsy