Western blotting signal problem - very weird and does not make any sense. (Aug/16/2006 )
Ok guys,
This is my problem. I have two blots. Both with same treatment grps but differnt time-points. I use the same antibody (prim and sec, I make it in one tube and split it 50/50) for the blots.
After I develop using ECL i place both my membranes inside the same packet one below the ohter and develop them. When I first expose it, both my bands show up. Then when I want to adjust the size of my bands and reexpose to a new film, the bottom blot hardly shows any signal or a faint one. The top one is just fine. This reexposure is say within 3-5 mins of the first exosure and 5-7 mins after the chemi.
I did this with two differnt antibodies (first with anti-rabbit and the second anti-mouse) The second one was for B-actin. Even this did not show up well. I have no idea whats happening.
I switched cassettes...still zilch...
any ideas
p..
does anyone have any bright ideas???
Someone (may be a lab midget) moved your blots during primary or secondary antibody incubation.
do you add fresh substrate before the second exposure?
ECL substrates are normally ok for 30'... and don't denature the blots...
I don't know what can happen...
I think the problem is with the bottom part of the film. Probably does not have an even coating or something. When I switched to a differnt box of film...everything was fine.
p..
Its hard to be a detective. Dont you agree, Casper? I am glad this is not a problem for you any more.
you are absolutely right my dear Watson
This is my problem. I have two blots. Both with same treatment grps but differnt time-points. I use the same antibody (prim and sec, I make it in one tube and split it 50/50) for the blots.
After I develop using ECL i place both my membranes inside the same packet one below the ohter and develop them. When I first expose it, both my bands show up. Then when I want to adjust the size of my bands and reexpose to a new film, the bottom blot hardly shows any signal or a faint one. The top one is just fine. This reexposure is say within 3-5 mins of the first exosure and 5-7 mins after the chemi.
I did this with two differnt antibodies (first with anti-rabbit and the second anti-mouse) The second one was for B-actin. Even this did not show up well. I have no idea whats happening.
I switched cassettes...still zilch...
any ideas
p..
Hi Casper,
I agree w/mdfenko. Just need a fresh ECL substrate mix.
The reason I say that is even when I come in the next day to reprobe my blot, the HRP in the secondary Ab still works robustly even if I let it sit overnite between plastic sheets in its old ECL mix without washing.
Ameer
It could be the ECL reagents, different companies have ECLs with different half-lives. I've tried several ones and the best is Amersham's (Pierce is also good)