Amount of antibody used in Western Blot - (Nov/23/2011 )

What is the minimum amount of diluted antibody solution one can use and what important tips are there to follow with small amounts of incubated antibody?

Does it make a difference if your membrane faces down or up during antibody incubation?

the minimum amount is the minimum that gives you complete coverage.

face up if using minimal amounts of antibody.

if you use larger volumes, so that the membrane can move freely in the plate, then it doesn't matter, as long as the membrane doesn't settle. i prefer face up at all times.