MAP and Jun kinase assays

Jim Woodgett jwoodget at oci.utoronto.ca
Tue Apr 2 11:58:07 EST 1996


In article <9604020315.AA00893 at uranus.med.pitt.edu>, corey at MED.PITT.EDU (Seth 
Corey) writes:

> new england biolabs sell kits. 

NEB sell antibodies to certain phosphorylated components of the MAPK and SAPK/
JNK pathways (which are pretty specific in our hands).  However, there are 
currently no "kits" for assaying activity (at least of the Jun kinases or p38/
CSBP/HOG).  This will likely change in the near future.

In the meantime, the assays are relatively simple if you can get hold of 
immunoprecipitating antibodies and the substrates (standard 32P incorporation 
assay).  Avoid peptide substrates as they are very insensitive.  In the case 
of the SAPK/JNK assays, you can do without antibodies and pull down the 
kinases from extracts with GST fusion protein substrates such as GST-Jun or 
GST-ATF2.  After washing the beads to remove non-specifically associated 
proteins just add radioactive ATP and the associated kinases will incorporate 
32P phosphate.

Jim Woodgett
Ontario Cancer Institute
Toronto

mailto:jwoodget at oci.utoronto.ca









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