Assays for Akt.
An increasing number of publications have underscored the importance of the serine/threonine kinase Akt in the regulation of cell survival, proliferation, and insulin-dependent metabolic cell responses. Critical to the understanding of Akt signaling in cells are experimental methods that assess its activation and phosphorylation state. In this chapter, we evaluate the most commonly used techniques to examine Akt activity. Immunocomplex kinase assays that utilize Akt-specific substrates are described, as is the use of phosphospecific antibodies directed against Akt phosphorylation sites. Furthermore, we introduce coupled enzyme assays that indirectly measure the activity of Akt by examining the activity of Akt substrates.
Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
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D000818:Animals; D002460:Cell Line; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection / Q000379:methods
D007527:Isoenzymes; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt