A chemiluminescent microtiter plate assay for sensitive detection of protein kinase activity.
A chemiluminescent protein kinase assay using biotinylated substrate peptides captured on a streptavidin-coated microtiter plate and monoclonal antibodies to detect their phosphorylation is described. Assay conditions were optimized and validated for sensitive measurement of protein kinase A, protein kinase C, Ca2+/calmodulin-dependent protein kinase II (CAM-KII), receptor interacting protein, and src activities. The newly developed chemiluminescent assay has several advantages over currently used radioactive or colorimetric methods. It is highly sensitive at low enzyme and substrate concentrations and high, close to physiological ATP levels. It is fast, simple to perform and amenable to automation and high-throughput drug screening. The assay is also robust, exhibiting minimum interference from solvents and test substances from various sources. Overall, among the presently available methods for the detection of protein kinase activity, chemiluminescence was found to provide the highest sensitivity under conditions most closely mimicking the intracellular environment. This assay is expected to be useful in both academic and industrial laboratories, especially in identifying novel classes of protein kinase inhibitors.
Lehel
C
C
Tularik Inc., South San Francisco, California 94080, USA.
Daniel-Issakani
S
S
Brasseur
M
M
Strulovici
B
B
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D003124:Colorimetry; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis; D008163:Luminescent Measurements; D051379:Mice; D010766:Phosphorylation; D011506:Proteins / Q000032:analysis; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D019061:src-Family Kinases / Q000032:analysis
D005904:Glial Fibrillary Acidic Protein; D011506:Proteins; D000255:Adenosine Triphosphate; D019061:src-Family Kinases; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C506251:Ripk1 protein, mouse; D017868:Cyclic AMP-Dependent Protein Kinases