stripping buffer! - didn't strip after 30min (Nov/28/2008 )
this was my first attempt to ever use any stripping buffer and it did not work out. to be honest I have no experience with it whatsoever. I used a commercial stripping buffer to strip my membrane after BCIP/NBT visualization but i couldn't remove the stain at all. I added 20ml to the membrane and left it on a rocker for 30 min but it did not strip my secondary antibody to give me a clear membrane to re-treat with another primary antibody.
how do you guys do it usually?
-Curtis-
QUOTE (Curtis @ Nov 28 2008, 09:18 PM)
this was my first attempt to ever use any stripping buffer and it did not work out. to be honest I have no experience with it whatsoever. I used a commercial stripping buffer to strip my membrane after BCIP/NBT visualization but i couldn't remove the stain at all. I added 20ml to the membrane and left it on a rocker for 30 min but it did not strip my secondary antibody to give me a clear membrane to re-treat with another primary antibody.
how do you guys do it usually?
how do you guys do it usually?
what is the temperature when you did your stripping? I usually did it at 50 oC
-stone757-
QUOTE (Curtis @ Nov 28 2008, 06:18 AM)
this was my first attempt to ever use any stripping buffer and it did not work out. to be honest I have no experience with it whatsoever. I used a commercial stripping buffer to strip my membrane after BCIP/NBT visualization but i couldn't remove the stain at all. I added 20ml to the membrane and left it on a rocker for 30 min but it did not strip my secondary antibody to give me a clear membrane to re-treat with another primary antibody.
how do you guys do it usually?
how do you guys do it usually?
BCIP/NBT is not to strip with classical stripping buffers as it is a precipitated colored substrate; stripping buffers are used to get rid off the antibodies;
I propose to strip the antibodies and then to bleach the blot (I would start with 3% H2O2)
-The Bearer-
QUOTE (The Bearer @ Nov 28 2008, 07:13 AM)
BCIP/NBT is not to strip with classical stripping buffers as it is a precipitated colored substrate; stripping buffers are used to get rid off the antibodies;
I propose to strip the antibodies and then to bleach the blot (I would start with 3% H2O2)
I propose to strip the antibodies and then to bleach the blot (I would start with 3% H2O2)
thanks The Bearer, but if I bleach the membrane I won't be losing any protein, right? will it just remove the antibodies and the stain?
stone757, I did it at RT
-Curtis-