[Date Prev][Date Next][Thread Prev][Thread Next][Date Index][Thread Index]

in gel kinase assays

There seems to be a fair degree of interest in the use of in gel kinase
assays and the literature contains many reports of the "successful" use of
the approach. However, I am frankly concerned about the specificity and
reliability of this approach. It has been used most commonly for the study
of MAP kinases and has typically employed the use of myelin basic protein
(MBP) as a substrate. MBP is an extremely promiscuous substrate. Even
highly specific kinases such as Mek1 have been found to phosphorylate MBP
in vitro. Furthermore, there seems to be many more MAP kinase-like isoforms
besides Erk1, Erk2, p38 Hog, SAPK/JNK in the 40 to 46 kDa range depending
on the species and tissue under examination. With as much as 2000 protein
kinases predicted to exist in the human genome, the assignment of a
radioactive band from an in gel assay to a specific kinase is rather a
dubious proposition at best. An approach that might circumvent some of my
concerns could involve first the specific immunoprecipitation of the kinase
of interest, and then the application of the in gel kinase assay approach.
This would markedly reduce the chance of a non-specific endogenous
substrate for a kinase in the kinase sample comigrating with that kinase on
the SDS-PAGE gel. Furthermore, the size of the radioactive band following
autoradiography should correspond to the predicted size of the target
kinase. This would further ensure that the antibody selectively
immunoprecipitated the target kinase and not an immunologically related
kinase.  Incidently, the inclusion of trimethylamine N-oxide or possibly
betaine could improve the rate and degree of renaturation of kinases from
the SDS-PAGE. These reagents are known to improve the renaturation of
urea-treated proteins in solution. I would be pleased to learn if anyone
has tried the in gel kinase assay on immunoprecipitated protein kinases and
compared the data to the results from the straight in gel kinase assay.

Steven Pelech, Ph.D.
Department of Medicine
Rm S125, 2nd Floor, Koerner Pavilion
2211 Wesbrook Mall
University of British Columbia
Vancouver, B.C.
Canada  V6T 1Z3

Office phone: (604) 822-8086
Office facsimile: (604) 822-8693
Confidential home facsimile: (604) 272-2102
E-mail: spelech@home.com