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MAP kinase assay reply



Hi Alex,

Phosphorylated  MBP migrates as a 20 kDa protein in a standard SDS 15%
Polyacrylamide gel.

Running a gel, followed by autoradiography is be much easier than the
filter assay.  For quantitation  use a phosphorimager.  But if it is not
available, it remains possible to scan the autoradiographs or including
stained protein markers in the running lanes and cut off pieces of gel to
be counted.

*** IMPORTANT :

1) Do NOT ALLOW the MIGRATION FRONT stained with the bromophenol blue to
come out the gel, it contains unreacted nucleotides which will pollute your
running buffer.
2) BEFORE FIXING and DRYING the gel: Chop off a large portion of the gel
above the migration front (If you do not do this step, unreacted ATP will
diffuse everywhere, contaminate your labware and make a high background.
Using a 15% polyacrylamide gel, MBP will migrate in the middle between the
front and the top of the gel.  You will easily check with a hand detector,
that the unreacted nucleotide front has been eliminated.

*** For cleaner results, you may want to load one lane out of two (MBP
diffuses a little)

Good luck,




Olivier Bensaude, DR - INSERM
Régulation de l'Expression Génétique
CNRS UMR 8541 - Ecole Normale Supérieure
46, rue d'Ulm -  75230 PARIS Cedex 05
Tel: 33 (0)1 4432 3410 / Fax: 33 (0) 1 4432 3941