A rapid and sensitive quantitative kinase activity assay using a convenient 96-well format.
Activation of protein kinases in response to growth factor and extracellular matrix stimulation has been implicated in regulating a number of cell functions including differentiation, gene expression, migration, and proliferation. An improved quantitative assay for measuring protein kinase activity is crucial to the detailed study of this important category of signaling proteins and their role in regulating cell behavior. We describe a modified in vitro kinase activity assay that is both sensitive and quantitative. It offers several advantages when compared to the traditional immunoprecipitation/kinase assay: (i) high sensitivity that reduces the required amount of cell lysate by an order of magnitude, (ii) an immunoseparation technique utilizing antibody immobilization onto the surface of microtiter wells that replaces the cumbersome immunoprecipitation method, (iii) a 96-well plate configuration that eases handling of multiple samples and increases throughput of the assay, and (iv) the use of 96-well filter plates that greatly reduces radioactive liquid waste generation. While we implement this technique in a case study for measuring the activity of extracellular signal-regulated kinase 2 (ERK2), this assay can be extended to studying other protein kinases by using an appropriate antibody and in vitro substrate for the kinase of interest.
Asthagiri
A R
AR
Department of Chemical Engineering and Division of Bioengineering & Environmental Health, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Horwitz
A F
AF
Lauffenburger
D A
DA
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis / Q000378:metabolism; D006224:Cricetinae; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D019950:Mitogen-Activated Protein Kinase 1; D011494:Protein Kinases / Q000032:analysis*; D012680:Sensitivity and Specificity
D004815:Epidermal Growth Factor; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1