Guanidinium Chloride - (Jul/03/2009 )

Hi all, I am using a stripping protocol with Guanidinium Chloride to strip my PVDF membranes. It is a very simple protocol and it works at another workgroup in another lab just fine. The problem is that my PVDFs become all black after stripping, I dont get any signal, just background.
Has anybody had any experience with Guanidinium Chloride? Any ideas of what might be going wrong with my system?

Thank you all so much

Aris on Jul 3 2009, 03:55 PM said:

have you reblocked before reprocessing the stripped blot?

yes i have reblocked again
for some reason guanidin chloride enhances the non specific binding of either the primary or the secondary

what are your blocking conditions? could increase the blocking concentration? or decrease the antibody concentration?

little mouse on Jul 16 2009, 12:58 AM said:

5% milk is my blocking media. The other workgroup does not block at all. I have tried that also. The strange thing is now that the fellow workign next to me in the lab has wonderful results. I have given him the protocol and it works just fine for him also. So i dont know what i am doing wrong

maybe the problem is not the stripping but the blocking. Are you using streptavidin for the detection? or anti-phospho antibodies?

little mouse on Jul 16 2009, 05:27 AM said:

We are not using streptavidin, we are not using phospho Abs. The other workgroups have used phosphoAbs but the other fellow here is not and he doesnt have any problem. It is deffinetely sth in my own system but i dont know what it is or how to trace it down.

are you using homemade or purchased antibody?

if homemade, what adjuvant did you use? is it the same or similar to your blocking agent?

maybe your blocking agent contains an epitope, similar to the antigenic epitope, that gets exposed when treated with gucl (may sound a little far-fetched, but stranger things have happened).

mdfenko on Jul 17 2009, 07:53 AM said:

It does not matter whether homemade or commercial. It happens always no matter what. The epitope theory is also my guess after all this time of experiments. My only solution is to cut the primary concentration down to half but this gives me very moderate results. Strange strange
strange...

the reason i asked if it was homemade or commercial is because you would know (or be able to find out easily) what the adjuvant was used, not so (or, at least, not as easily) with commercial.

if the adjuvant was similar to the blocking agent then your antibody may be to that (could be with polyclonal).