how to get red of high secondary signal (View forum version)



michalcn

Posted 02 July 2011 - 09:41 AM

hi,
i'm doing western and want to reprobe the membrane with different secondary ab the problem that I still get the previous signal also, I heard that you can block the signal using ponceau instead of stripping,do anyone know protocol?

BioMiha

Posted 02 July 2011 - 11:29 AM

ponceau is just a dye to stain proteins on the membrane, I have never heard it could actually strip it. I think you are better of redoing your western blot.

bob1

Posted 02 July 2011 - 02:32 PM

Are you using HRP or alkaline phosphatase as the secondary conjugate? If HRP, you should be able to use Sodium Azide to permanantly block the activity of the first secondary conjugate.

michalcn

Posted 05 July 2011 - 04:02 PM

Are you using HRP or alkaline phosphatase as the secondary conjugate? If HRP, you should be able to use Sodium Azide to permanantly block the activity of the first secondary conjugate.


Basically I was trying avoid stripping...
yes, I do use HRP so do you have the protocol of blocking the first activity?
THANKS a LOT

bob1

Posted 05 July 2011 - 06:59 PM

Basically just add some azide (i'd go for about 0.1% w/v) to some PBS and incubate for a couple of hours. In theory this should work, though I have never tested it myself. Azide is used to block endogenous peroxidase in IHC.