how to check protein-primary antibody binding - Is there any method or technique to check or proof read the primary Ab (May/02/2007 )

Hi Is there any method or technique to check/proof read the primary Ab-protein binding. ive checked the profile through staining and also the transfer. my developer and fixer are also ok.

however im unable to check if my primary Ab-protein are binding
and primary Ab-secondary Ab binding.

i sometimes get a weak signal but mostly my film is blank!!!!

i have muscle samples and my protein is abt 40-50kDa.

is there any method to check whether my protein of interest is degraded or not in sample.???

NEED SUGGESTIONS

to check that your secondary is binding, you can dot plot the primary.

To check Pr-primary Ab binding, I suppose you can use something like the BIACORE affinity test, but I am afraid that it might be expensive. Your positive control worked? Have you try load your interested protein pure (like GST-fusion protein or so, with different concentrations, together with controls, of course) and see if your primary Ab can recognize those (can try Ab dose there, too). That way you can know that it is not because of degradation of your protein. If the affinity is a little weak, try washing less or more gently or less stringent (less Tween20)?

put a drop (0.5 - 1 µL) of antibody on dry nitrocellulose membrane, let it dry, then you perform a western-blot as usual (blocking, incubation with secondary antibody, wash....).

if your sample is not very expensive or time consuming to prepare, you may do a gel in which you load increasing amounts of proteins. Then you may see how much total proteins you need to load.
Basically, overnight-4°C incubation give better results. Then you may do shorter blocking (for some AB i don't block and incubate O/N the 1st AB).

What is your concentration of AB : you may increase
how much tween do you add ? try 0.025%
try PBS-T instead of TBS-T
Did you try BSA as blocking instead of milk ?