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.edu<mailto:mach...@med.unc.edu>
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