Preparation of anti-phosphoserine and anti-phosphothreonine antibodies and their application in the study of insulin- and EGF-induced phosphorylation.

We prepared antibodies against phosphoserine (P-Ser) and phosphothreonine (P-Thr) by immunizing rabbits with P-Ser or P-Thr conjugated to bovine serum albumin. The antibodies (anti-P-Ser and anti-P-Thr) were purified using P-Ser or P-Thr affinity columns. Anti-P-Thr was highly specific for P-Thr, while anti-P-Ser showed weak cross-reactivity with P-Thr. We showed that these antibodies can immunodetect serine/threonine phosphorylated insulin and epidermal growth factor (EGF) receptors and several proteins which are phosphorylated on serine/threonine residues in response to insulin or EGF stimulation. The antibodies will certainly provide a good tool for discovering novel kinases and substrates involved in signal transduction.