Dye-binding protein assay giving very low signals - any ideas?

Achim Recktenwald, PhD achim at ibex.ca
Wed Mar 19 08:23:57 EST 1997


David wrote:
> 
> I'd be very grateful for any advice on this problem:
> 
> I've eluted some polyclonal antibodies from an antigen column, and
> dialysed them into a suitable buffer - the strange thing is that
> although the absorbance at 280nm indicates that the proteins are at a
> certain, relatively high level (e.g. 1mg/ml), when using the Bradford
> assay (the microassay procedure, using the solution from Bio-Rad), one
> gets a very low absorbance at 595nm, indicating a concentration about 2
> orders of magnitude less (a positive control with BSA works fine).
> 
> I've run the antibodies on an SDS-PAGE gel, and there's definitely
> antibody there (they also work on Western blots), but it's a bit
> disconcerting that the dye-binding assay doesn't work with these
> antibodies.
> 
> I know that certain levels of certain substances can interfere with the
> assay, but these should not be present in these solutions (which have
> been dialysed into Tris-buffered saline solutions).
> 
> Any ideas as to what may be causing the problem? Has anyone had similar
> problems?
> 
> Thanks in advance for any help you can give with this,
> 
> David
> 
> Dept. of Biochemistry,
> University of Cambridge
> England
> 
> das1002 at cam.ac.uk


Hi David,

What is your buffer matrix?  Do you have a high level of DTT,
beta-mercaptoethanol, or a high amount of detergent in your
elution-buffer?  Check the booklet coming with the assay-solution from
Biorad; there should be a listing of interfereing substances.

Achim

_______________________
Achim Recktenwald, PhD.
IBEX Technologies, Inc.
Biochemistry
5485 rue Pare
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Canada

achim at ibex.ca


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