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.
Kono
S
S
Department of Internal Medicine, Kyoto University, Japan.
Kuzuya
H
H
Yamada
K
K
Yoshimasa
Y
Y
Okamoto
M
M
Nishimura
H
H
Kosaki
A
A
Inoue
G
G
Hayashi
T
T
Imura
H
H
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis* / Q000276:immunology; D010769:Phosphothreonine / Q000032:analysis* / Q000276:immunology; D011817:Rabbits / Q000276:immunology; D011972:Receptor, Insulin / Q000378:metabolism*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
D000906:Antibodies; D007328:Insulin; D010710:Phosphates; D010750:Phosphoproteins; D010769:Phosphothreonine; D010768:Phosphoserine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011972:Receptor, Insulin; D013755:Tetradecanoylphorbol Acetate