Blocking before hybridizations

[Monique]

I was wondering how the blocking of a sample works with serum before hybridizing it with an antibody.

I mean, you want to block everything aspecific so that the antibody doesn't bind everthing, but with that it blocks things that the antibody is supposed to bind right?

Or is it all about affinity, the antibody should have enough affinity to bind no matter that it is blocked..

And exactly what is the blocking agent in serum? Albumin? And why does it block?

[:D]

I'm braindead after waking up at 6 and coming home at 7, eating and after that studying and then going to sleep around midnight..

[iansmith]

The blocking solution is made with a blocking agents which is could either be serum albumin, skim milk or tween. The blocking agent will cover the protein or surface with a slight coat and block unspecific binding but it will not influence the specific binding.

I think the pH, salt concentration and concentration of blocking agent will influence the stringency of the binding. pH and salt concentration probably need to be stable because buffers are always used.