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JNK Kinase Assay

JNK Kinase Assay

 

THE BASIC PROTOCOL IS THE SAME AS THE MAP KINASE ASSAY, with the following changes:

 

REACTION BUFFER

25mM HEPES pH 7.4

25mM b-glycerophosphate (not needed)

50nM OA

25mM MgCl2

0.1mM Na3VO4

0.5mM DTT

 

Kinase assay: 10ml beads

                        26ml Reactionbuffer

                        2ml GST-Jun (5mg/ml)

                        1ml 1mM ATP

                        1ml [g32P]-ATP (10mCi) (5mCi is OK)

 

Incubate 15 minutes at Room temp

Stop with SDS sample buffer

SDS PAGE and autoradiography