A generic high-throughput screening assay for kinases: protein kinase a as an example.
A generic high-throughput screening assay based on the scintillation proximity assay technology has been developed for protein kinases. In this assay, the biotinylated (33)P-peptide product is captured onto polylysine Ysi bead via avidin. The scintillation signal measuring the product formation increases linearly with avidin concentration due to effective capture of the product on the bead surface via strong coulombic interactions. This novel assay has been optimized and validated in 384-well microplates. In a pilot screen, a signal-to-noise ratio of 5- to 9-fold and a Z' factor ranging from 0.6 to 0.8 were observed, demonstrating the suitability of this assay for high-throughput screening of random chemical libraries for kinase inhibitors.
Mallari
Rommel
R
Lead Discovery Department, AGY Therapeutics, Inc., 290 Utah Ave., South San Francisco, CA 94080, USA.
Swearingen
Elissa
E
Liu
Wei
W
Ow
Arnold
A
Young
Stephen W
SW
Huang
Shu-Gui
SG
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001360:Avidin / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D001710:Biotin / Q000378:metabolism; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011107:Polylysine / Q000378:metabolism; D015203:Reproducibility of Results; D012588:Scintillation Counting
D010455:Peptides; D001360:Avidin; D011107:Polylysine; D001710:Biotin; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D004121:Dimethyl Sulfoxide