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

Dr E. Buxbaum EB15 at le.ac.uk
Thu Mar 20 05:21:49 EST 1997

das1002 at cam.ac.uk (David) wrote:

>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).

Serum albumin has a very high binding capacity for Coomassie stain and therefore gives 
artificially high readings in the Bradford assay. You can either use IgG protein standards 
(commercially available for example from Pierce) or use a different protein detection method 
like Lowry or BCA. A280 should also work, provided you have no other absorbing substances 

Note that in general the standard protein should be as similar as possible to the protein to be 
measured, IgG is therefore more suitable as standard for antibody determination than BSA.

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