Michael Thompson <[log in to unmask]> 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" <[log in to unmask]>
> To: [log in to unmask]
> 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
>
> [log in to unmask]
>
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