first and 2o Ab with or without blocking solution - what do you usually use? (Nov/13/2007 )

Which is better.

I’m almost new doing western (I just did it twice when I was a student).

I remember we blocked, added the first Ab, wash and then 2o Ab or somethink like that.

The guy was working here dilute both first and 2o Ab in blocking solution.

The new guy add the fist Ab (without blocking solution) and the 2o one with blocking solution.

What is the best and why???????????

I always wanted to just do 1o and 2o together in blocking solution with no initial blocking (it's just an equilibrium thing right?), but I always block overnight, 1o overnight, then 2o for an hour (with washes in between). Is it just superstition? Like I have a week or two to dedicate to the question - I guess it would actually be worth my time in the long run if I could cut out all the work. Anypone ever tried comparing such protocols?

I always do mine in blocking solution.

I do mine in blocking solution, unless if I see a weak signal, then I switch to wash buffer.
Depends on the antibody.

I use 5% no fat dry milk blocking solution for 1 hour RT, than, remove this solution and replace with 3% milk solution with my Abs. between Ab1 and Ab2 you must wash the membrane

Hi princess,
always it is better to dilute the antibodies in blocking solution so that non-specific binding is minimized
as you are diluting the antibody in blocking solution any non-specific binding will happen in the blocking solution itself which will be washed away easily
all the best
bye
Leelaram

If you have a very weak signal, so, a not so good working antibody, don't use milk or try BSA, otherwise I always use milk in both for background reduction.

thank you very much for your anwers, I will try it this way.

as you say, if I have no signal, I will try BSA.

Wish me luck!