Problems after stripping Western blot - (Jan/08/2008 )
Hey there. I hope someone can give me some advice. My lab typically performs AP conjugated - precipitation Westerns but my current project relies on pretty low levels of protein which aren't being detected by AP conjugated secondary ABs. Thus, I've made the switch to HRP conjugated secondaries for better detection and membrane stripping. The initial protein detection by ECL Plus and radiography is fine. However, after stripping the membrane with Pierce Restore Stripping buffer and reprobing for the same or different antibodies, the background on the film goes through the roof! I am currently stripping for ~15 min, wash 3 x 10min TBS-T, verify the HRP has been removed with ECL substrate / film, block 30 - 60min 10% milk then add primary antibody diluted in milk O/N at 4 degrees. I've tried reducing secondary antibody concentration, increasing blocking time, increasing washed, all to no avail. Has anyone observed anything similar? Thanks so much.
fmusc
I do two more wash after the stripping, quick washes in large volume, just to really dilute and eliminate the stripping buffer, and then do 3 washes of 10 minutes. I don't know if it could explain your problem.
Maybe you could also try to incubate the first Ab 1-2 hours at RT instead of ON, to reduce background?
I have tried using the AB at RT instead of O/N and achieve the same high background. Next time I'll try more washes after stripping but the volume I'm using should be sufficient. Thanks for the post.
Maybe you could also try to incubate the first Ab 1-2 hours at RT instead of ON, to reduce background?
i gived a try to block after stripping with BSA.
It's possible that your secondary antibody is too concentrated too
I have always blocked after stripping, 1 hr with either 5% BSA or 10% milk, same result. Diluting the secondary 1:20,000 from previous 1:2000 also has no effect. Any other ideas? Thanks.
It's possible that your secondary antibody is too concentrated too
It's possible that your secondary antibody is too concentrated too
it is not necessary to block again after stripping if you wash in TTBS; reduce your block concentration for the initial blocking (10% milk is really high); if you use polyclonal antibodies, unspecific cross reactions are not unusual; use an alternative antibody to be sure of the specificity of the signal or try to work with a blocking peptide