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

David das1002 at cam.ac.uk
Tue Mar 18 06:24:48 EST 1997


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



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