IMMUNOBLOTTING - NEED HELP

Henk Veldman H.Veldman-2 at neuro.azu.nl
Fri Jun 1 03:00:47 EST 2001


L R schreef:

> Hello,
>
> I am getting a lot of unspecific Ab binding doing a western.  If I am
> to use serum in the blocking buffer with the Ab, would it be the serum
> of the organism the Ab was raised in

If this antibody binds to your membrane (as it is supposed to do) guess
what would happen in the next step?

> or the opposite serum (ie. for
> the primary Ab solution, the serum of the organism that the secondary
> Ab was raised in and vice-versa)?

For the primary antibody: yes.

For the secondary antibody: heavens no, it was raised to the primary so it
will be neutralized by it!
Generally there should be no need to do anything special with the
secondary antibody as long as it is of good quality and used in the proper
way. Problems with second and later steps can be identified by processing
a blot while skipping the earlier steps.

Some of the most important things to avoid background:
    Block  your membrane effectively before using any antibody
    Use every antibody in an optimal (highest usable) dilution
    Use effective washing

Henk
--
H. Veldman
Laboratory for Experimental Neurology (NMZ)
University Medical Center Utrecht (AZU)





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