A solid-phase assay for the determination of protein tyrosine kinase activity of c-src using scintillating microtitration plates.
A solid-phase assay for the determination of protein tyrosine kinase (PTK) activity has been developed. The transfer of 33PO4 from ATP to the synthetic substrate poly(Glu, Tyr) 4:1 attached to the bioactive surface of scintillating microtiter plates served as the basis to evaluate enzyme activity. The procedure eliminates detection with phosphotyrosine antibodies, tedious separation of phosphorylated peptides with phosphocellulose membranes, and extensive washing steps. For these reasons, the traditionally time-consuming procedure can be performed with a simple three-step protocol. The method is highly accurate, rapid, and robotics friendly. The advantages over existing assays make this procedure especially suited for high throughput applications.
Braunwaler
A F
AF
Ciba-Geigy Corporation, Summit, New Jersey 07901, USA.
Yarwood
D R
DR
Hall
T
T
Missbach
M
M
Lipson
K E
KE
Sills
M A
MA
D000818:Animals; D001331:Automation; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D007700:Kinetics; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D017027:Protein Tyrosine Phosphatases / Q000032:analysis* / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000378:metabolism*; D011934:Reagent Strips; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D015203:Reproducibility of Results; D012588:Scintillation Counting / Q000379:methods; D012680:Sensitivity and Specificity; D018411:Spodoptera; D013379:Substrate Specificity; D014162:Transfection; D019061:src-Family Kinases
D010710:Phosphates; D010761:Phosphorus Radioisotopes; D011934:Reagent Strips; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; D017027:Protein Tyrosine Phosphatases