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
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#2
mdfenko
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Posted 06 July 2009 - 08:28 AM
Aris, on Jul 3 2009, 03:55 PM, said:
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
Has anybody had any experience with Guanidinium Chloride? Any ideas of what might be going wrong with my system?
Thank you all so much
have you reblocked before reprocessing the stripped blot?
talent does what it can
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genius does what it must
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#3
Aris
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Posted 15 July 2009 - 10:01 AM
yes i have reblocked again
for some reason guanidin chloride enhances the non specific binding of either the primary or the secondary
for some reason guanidin chloride enhances the non specific binding of either the primary or the secondary
#4
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Posted 16 July 2009 - 12:58 AM
what are your blocking conditions? could increase the blocking concentration? or decrease the antibody concentration?
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Posted 16 July 2009 - 04:14 AM
little mouse, on Jul 16 2009, 12:58 AM, said:
what are your blocking conditions? could increase the blocking concentration? or decrease the antibody concentration?
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
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Posted 16 July 2009 - 05:27 AM
maybe the problem is not the stripping but the blocking. Are you using streptavidin for the detection? or anti-phospho antibodies?
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Posted 16 July 2009 - 09:23 AM
little mouse, on Jul 16 2009, 05:27 AM, said:
maybe the problem is not the stripping but the blocking. Are you using streptavidin for the detection? or anti-phospho antibodies?
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.
#8
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Posted 17 July 2009 - 07:53 AM
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).
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).
talent does what it can
genius does what it must
i do what i get paid to do
genius does what it must
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#9
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Posted 18 July 2009 - 05:05 AM
mdfenko, on Jul 17 2009, 07:53 AM, said:
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).
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).
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...
#10
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Posted 20 July 2009 - 11:10 AM
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).
if the adjuvant was similar to the blocking agent then your antibody may be to that (could be with polyclonal).
talent does what it can
genius does what it must
i do what i get paid to do
genius does what it must
i do what i get paid to do
#11
little mouse
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Posted 20 July 2009 - 11:31 PM
do you use the same antibodies than your colleagues that succeed the stripping?
they must do something different. Do they use the same membranes (PVDF, nitrocellulose, from the same or from a different company)? I don't understand why you have black background, but the clue is in the little difference in the protocol of your colleagues.
they must do something different. Do they use the same membranes (PVDF, nitrocellulose, from the same or from a different company)? I don't understand why you have black background, but the clue is in the little difference in the protocol of your colleagues.
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Posted 22 July 2009 - 04:52 PM
little mouse, on Jul 21 2009, 12:31 AM, said:
do you use the same antibodies than your colleagues that succeed the stripping?
they must do something different. Do they use the same membranes (PVDF, nitrocellulose, from the same or from a different company)? I don't understand why you have black background, but the clue is in the little difference in the protocol of your colleagues.
they must do something different. Do they use the same membranes (PVDF, nitrocellulose, from the same or from a different company)? I don't understand why you have black background, but the clue is in the little difference in the protocol of your colleagues.
We use the same PVDFs all of us. The only difference is that my Abs come in preserved in NaN3 whereas the others come in PBS. I really dont know if the NaN3 is making all the problem.
The day b4 yesterday i stripped and exposed to I Ab in 1:6000 concentration. All back. After that I stripped again, checked just with the secondary for signal (no signal so ok) and i exposed the blot to the same primary only the half concentration (1:12000).
Guess what... significant improvement...not perfect but conciderably better...
One other fellow did for me side by side the same with his membrane and my Abs...all black
So....what the h... is happening..is it possible that the NaN3 is causing te trouble? ANd if so, how can i avoid it? All my Abs are in NaN3
