Why is there high background and/or diminished signal with anti-phosphotyrosine antibodies?
Check the blocking solution that you are using. Non-fat dry milk, which we recommend for all blots EXCEPT phosphotyrosine blots, has phosphate-containing proteins that bind to the membrane. By blocking with this reagent, potential epitopes are present all over the blot. Diluting the anti-phosphotyrosine antibody in this solution will occupy many of the antibody binding sites, leaving fewer antibodies available for detection. So, use 1% BSA in TBS + Tween as the blocking solution – do not use milk.
