Stripping problems

[marnedo]

Hi,

I have some problems with the western blot when I want to probe other antibody. When I make the western for my protein there aren't problems and I obtained a really strong signal, but when we reproble the membrane for the control antibody there isn't any signal.

My stripping protocol is:
SDS 2%
Tris-HCl 0.5M pH 6.8
beta-mercapto 0.1%

I put my membrane with this buffer during 30 min at 60 ºC.
Then I wash with PBS-tween during 1 h changing the buffer.
I reblocked the membrane with PBS-tween-milk and incubate with the primary antibody.

This protocol worked in the past and I don't know where the problem is now.

Some idea??

Thanks.

María

[protolder]

Hola María, My stripping buffer is 50mM Tris 6.8 , the rest as yours and I incube the membrane with agitation during 20 min at 50ºC and incubating afterwards with secondary I haven´t any residual signal. Try this conditions if you want, because I haven´t any explanation for your lost of signal with the control ab. Good luck

[gregmey0]

You should check if it works with your control antibody on new membrane, maybe a problem with this one and not the strip ! Or redo the same antibody after stripping and see if it s work !

[marnedo]

Thanks, I'll try that and I hope to be lucky!