Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -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.
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.
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.
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.
. 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.
'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.
, 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
