Matt Hudson meh2 at le.ac.uk
Wed Aug 28 05:13:00 EST 1996

If you think it might be the secondary causing the trouble:

I got rid of my background problems by introducing a second blocking 
step. If you just block before incubation with your primary, try a 
secomd block in a lower conc. of blocking agent (say 1/5 what you used 
when blocking before the primary) before you probe with the secondary.

Also, we use powdered skimmed milk as the blocking agent, which is a 
whole lot cheaper than BSA (although would obviously cause problems with 
detection of some mammalian proteins)

Steve <smiles at bgsm.edu> wrote:
>Hey all!
>I'm doing a normal western blot, but our polyclonal antibodies seem to be 
>binding to many of the E. Coli proteins. It is possible that the 
>antibodies were raised to our protein, as well as any bacterial proteins 
>present in the blood during an infection in the rabbit.
>Is there any protocol to add cell extract to the BSA blocking buffer, or 
>the primary antibodies, which will prevent all this binding?
>Or is there a better way to block?

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