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

2011-06-17 Thread Arnon Lavie
To add to this discussion: Many of the comments to my question that started this thread do not sufficiently differentiate between accuracy and precision. While we all want an assay that is internally consistent (i.e., high precision), we do care a lot about accuracy (the degree of closeness of

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

2011-06-16 Thread Arnon Lavie
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

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

2011-06-16 Thread Filip Van Petegem
Dear Arnon, the Bradford method is not recommended for accurate measurements. The readings are strongly dependent on the amino acid composition. A much better method is using the absorption at 280nm under denaturing conditions (6M Guanidine), and using calculated extinction coefficients based

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

2011-06-16 Thread Oganesyan, Vaheh
: 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

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

2011-06-16 Thread aaleshin
@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

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

2011-06-16 Thread Francis E Reyes
Never had problems with evaporation (and this is in the relatively dry climate of Denver, CO, especially in the winter when the relative humidity is in the low 20%). Using the Thermo Scientific Nanodrop 2000c. We use it also as a prerequisite for ITC, which can be very sensitive to proper

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

2011-06-16 Thread Bosch, Juergen
@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

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

2011-06-16 Thread Filip Van Petegem
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

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

2011-06-16 Thread Quyen Hoang
We also have not experienced any problems with a Nanodrop 2000C. No one in my touched the two boxes of Bradford and BCA kits that we have, because we have been very happy with the Nanodrop. Quyen ___ Quyen Hoang, Ph.D Assistant Professor Department of Biochemistry

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

2011-06-16 Thread Bjørn Panyella Pedersen
On 2011-06-16 13:06, Filip Van Petegem wrote: Even if evaporation is not an issue, one has to take pipetting errors into account when dealing with small volumes. The relative error on 1-2ul is a lot bigger than on 50ul. True, but the nanodrop works independent of volumes, since it has a

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

2011-06-16 Thread aaleshin
: [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

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

2011-06-16 Thread David Briggs
I'll give my backing to the Nanodrop as well. I've used it in two different labs, for general yield checking use as well as prior to ITC experiments, and haven't found there to be any issues. That said, I've also used cuvettes, and I find that one the whole, cuvette-derived and nanodrop-derived

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

2011-06-16 Thread Tommi Kajander
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

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

2011-06-16 Thread Prince, D Bryan
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

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

2011-06-16 Thread Chun Luo
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

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

2011-06-16 Thread Justin Hall
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

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

2011-06-16 Thread Edward A. Berry
Arnon Lavie wrote: ~~~ We have been considering buying a Nanodrop machine (small volume, no dilution needed, fast, easy). However, while testing our samples using a colleague's machine, we have gotten readings up to 100% different to our Bradford assay (all fully purified proteins). For example,

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

2011-06-16 Thread Filip Van Petegem
] 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

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

2011-06-16 Thread David Briggs
'Fine' for me means comparable to SEC-MALLS measurements and reproducible. I use the E calculated from the sequence using the protparam server at Expasy. David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester

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

2011-06-16 Thread Jacob Keller
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

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

2011-06-16 Thread Petr Leiman
Totally support the statements below. We have had several proteins with A280 absorbance of 0.1 or less (at 1 mg/ml). You _have_ to use Bradford in the Nanodrop or whatnot to measure the concentration. Before purchasing the Nanodrop we used a Hellma TrayCell and a normal UV/Vis instrument.

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

2011-06-16 Thread Machius, Mischa Christian
With respect to the Edelhoch method and the ProtParam server, I would strongly recommend determining extinction coefficients experimentally and not rely on the ProtParam values. The reason is that the underlying extinction coefficients in the formula used by ProtParam and referenced there are

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

2011-06-16 Thread Oganesyan, Vaheh
[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

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

2011-06-16 Thread aaleshin
Mischa, You intrigued me. What is the experimental technique for the Extinction Coefficient measurement (which requires knowledge of protein concentration)? Let me guess, Bradford? Protein evaporation and weighing? Alex On Jun 16, 2011, at 4:22 PM, Machius, Mischa Christian wrote: With

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

2011-06-16 Thread aaleshin
Sorry for misprint, I meant evaporating water from a protein solution... On Jun 16, 2011, at 4:45 PM, aaleshin wrote: Mischa, You intrigued me. What is the experimental technique for the Extinction Coefficient measurement (which requires knowledge of protein concentration)? Let me guess,

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

2011-06-16 Thread Filip Van Petegem
A convenient fast way is the earlier mentioned Edelhoch method, as described in this paper which is referenced on the popular Protparam tool: http://onlinelibrary.wiley.com/doi/10.1002/pro.5560041120/pdf Filip On Thu, Jun 16, 2011 at 4:45 PM, aaleshin aales...@burnham.org wrote: Mischa, You

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

2011-06-16 Thread Shaun Lott
Just to add my 2c worth... The department here has a couple of nanodrops as a shared facility, one for DNA/RNA and one for protein. It has been noticeable that over time people has been getting decreased reliability of measurements on the latter machine cf cuvette measurements, presumably due

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

2011-06-16 Thread Scott Pegan
Here is also a very effective method: 1Gill, S. Hippel, P. v. Calculation of protein extinction coefficients from amino acid sequence data. Analytical Biochemistry 182, 319-326, (1989). On Thu, Jun 16, 2011 at 5:56 PM, Filip Van Petegem filip.vanpete...@gmail.com wrote: A convenient

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

2011-06-16 Thread Machius, Mischa Christian
The method is that by Edelhoch, mentioned a couple of times already in this discussion. It's also described in the paper by Pace et al., the same paper that the formula in ProtParam is from (ProtParam does not use the values determined by Gill von Hippel). Last time I looked into this, the

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

2011-06-16 Thread Pascal Egea
I would like to add something about the NanoDrop versus NanoPearl, I don't think that the path length is fixed on this instrument (the NanoDrop) since if I recall well, the instruments sets the path length as it scans through the droplet, hence the characteristic clicky noise that you hear as the

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

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

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

2011-06-16 Thread aaleshin
I see, by the experimental determination of the extinction coefficient you mean correction for the difference between unfolded (which can be computed accurately) and folded proteins. Am I right? Sorry for making this topic viral... Alex On Jun 16, 2011, at 5:06 PM, Machius, Mischa Christian

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

2011-06-16 Thread Dima Klenchin
The method is that by Edelhoch, mentioned a couple of times already in this discussion. You recommended determining extinction coefficients experimentally. How is plugging number of specific residues into a formula constitute experimental determination? It's also described in the paper by

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

2011-06-16 Thread Machius, Mischa Christian
On Jun 16, 2011, at 8:30 PM, Dima Klenchin wrote: You recommended determining extinction coefficients experimentally. How is plugging number of specific residues into a formula constitute experimental determination? That is a deeply philosophical question! Eventually, you'll be plugging in

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

2011-06-16 Thread Chelsy Prince
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

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

2011-06-16 Thread Richard Edward Gillilan
Hi Chelsy, yes we had a lot of trouble with the nanoview during that run. Even after going through the calibration procedure with the special fluid provided, we still had inconsistent results even on standards. Finally, I carefully cleaned the return light path of the instrument (a separate