Check the blocking solution that you are using. Non-fat dry milk suit for all blots except phosphor tyrosine blots, which has Phosphorylated proteins that bind to the membrane. By blocking with this reagent, potential epitopes are present all over the blot. Diluting the anti-phosphor tyrosine antibody in this solution will occupy many of the epitopes, leaving fewer antibodies available for detection. We suggest to use 1% BSA in TBS + Tween 20 as the blocking solution - do not use milk.
