Michael Thompson <[email protected]> a écrit :
There is a very simple and very quick method that yields an answer
approx. 15% reliable: measuring the increment of index of refraction
due to the protein. The measurement of an index of refraction can be
very accurate. You "only" need something like a 5µl drop at 1 mg/ml
(the order of magnitude should be correct...). Unfortunately, a
refractometer is not common in biology labs, but this is a very
valuable method.
The link between the increment of index of refraction and the protein
conc. can be found easily on the web.
Philippe Dumas
It is not surprising that your bradford and BCA assays don't agree
if you have no aromatic amino acids in your protein. Bradford dye
binds to hydrophobic residues, mainly aromatics, so I would guess
your bradford is consistantly giving lower measurements than the BCA
assay. I also wouldn't be surprised if the results of your Bradford
vary significantly between replicates. The BCA assay reagent
interacts with the backbone amides, not with any sidechains, so I
would tend to believe that measurement more than anything else you
have done.
I work with a protein that has very few hydrophobics (only one
aromatic - a Phe) and I have found that Bradfords are unreliable,
but the BCA assay tends to be consistent.
Mike
----- Original Message -----
From: "Arpit Mishra" <[email protected]>
To: [email protected]
Sent: Saturday, April 9, 2011 2:52:21 AM GMT -08:00 US/Canada Pacific
Subject: [ccp4bb] how to quantitate protein which dont have ne
aromatic residue
hello everybody
i am working on the protien which dont have any aromatic residue i
do fplc other purification using 220 absorption, but i want to
quantitate protein precisely i have tried using BCA nd bradford but
both methods quantification is not matching,,so any one is having
sum idea how to quantitate it precisely
thanks in advance for your valuable suggestion..
Arpit Mishra
--
Michael C. Thompson
Graduate Student
Biochemistry & Molecular Biology Division
Department of Chemistry & Biochemistry
University of California, Los Angeles
[email protected]
