western blot

[DSF]

Try using the primary antibody at no greater than 1:1000 
dilution, then wash afterward in TBST (0.1% Tween, though I'm not 
sure that the percentage of Tween matters) 1x20min, then 4x5min 
before you add the secondary. I use a donkey anti-rabbit from 
Calbiochem at 1:10,000 dilution. For detection, I use 
chemiluminescence, specifically Pierce's Ultraluminescence 
reagent (it's fairly new).
Lynette