Western blot stripping problem
Posted 26 January 2005 - 09:44 AM
How can I remove Ab 1st stained from my membranes? I have ised strip with NaOH 0.1M.
Posted 26 January 2005 - 10:31 AM
Posted 31 March 2005 - 05:30 AM
Usually, after I do the 1st stripping, all most proteins in the membrane disappeared ( I checked it by staining that membrane with Ponceau solution) or just remained a little.
The contents of stripping buffer I used is:
+ 0.2 M Glycine (pH 2.5)
+ 0.05% Tween 20
I incubated the membrane at 80oC for 30 minutes. And then, I washed it 10 min x 3 times with TBST before blocking with skim milk.
What's wrong in my method and how can I solve the problem?
Edited by Gin, 31 March 2005 - 05:37 AM.
Posted 31 March 2005 - 06:48 AM
i start to see same problems as you and i use the same protocol of stripping. Hence i'm wondering is "our" glycine too old? mine is more than 3 month...
Thanks for your reply.
Well, but I don't think so. Because another person who taught me that method, he also used the same Glycine with me. And of course, no matter Thus, I considered the pH of buffer, or the incubate-temperature, or the shaking... are they important to the result of stripping?
When you had that problem, how can you overcome it, Fred? You changed with "new" glycine?
Posted 31 March 2005 - 07:58 AM
i think ph is not that important caus i use pH 2.7 at 80°C for 30'
but the two last stripping were not by shaking i must admit, and the residual bands apperead just in the pas times.
hence i assume that shaking would increase efficiency