No signal of my normalisers - Western blot (Sep/08/2005 )
Dear everybody,
Please help me out.
I am doing an experiment where i use B actin as the normalizer. The problem is after i have stripped the filters then some of the wells did not have the B actin signals. Even if the intensity of the target protein in that well had the same strength as the others, so this could not be a loading problem, could it? What is wrong? I bought the mouse monoclonal antibody against human B actin from Santa Cruz Biotechnology, Inc. Has anyone used this before? I used 500X dilution. Should i reduce the dilution factor? Do you know any well known manufacturers for selling good antibody against human B actin?
Thanks!
I assume you are talking about a Western blot?
At which size is your target protein running compared to actin? It is very well possible that you had some are bubbles between your gel and the blot-membrane during blotting.
At which size is your target protein running compared to actin? It is very well possible that you had some are bubbles between your gel and the blot-membrane during blotting.
hi theo,
Yes, i am talking about western blotting. The size of my target is much higher than b actin. Do you know the reason for it?
So airbubbles could explain the local absence of actin staining?
Can you attach a picture of your result so that we see how it looks?