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kinases Immunoprecipitation kinase assay for PKA?
I have been measuring PKA activity in cells after sonication in a buffer
that contains no detergent. After sonication, the lysates are cleared by
centrifugation, leaving a substantial pellet of insoluble material. The
supernatant is then assayed for PKA activity (four reactions: -/+ Kemptide,
Has anybody ever successfully assayed PKA from cells lysed in a
detergent-containing buffer (e.g. NP40 or Triton X100 at 0.5 to 1%)? Is
there a way to IP PKA and then assess its activity, like is done for myriad
other kinases (e.g. ERKs, CDKs, RSKs,...)?
Any insights are greatly appreciated.
Alan K. Howe, Ph.D.
University of North Carolina at Chapel Hill
Lineberger Comprehensive Cancer Center
Dept. of Pharmacology, CB# 7365
Mary Ellen Jones Bldg, Rm 920
Chapel Hill, NC 27599-7365
(919) 966-5640 (FAX)