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ERK kinase assay
I have a little problem with the substrate MBP.
I i.p with ERK1 (Santa Cruz) and then perform the kinase assay using MBP (Sigma)
as substrate together with P32-ATP. Then I run a 15% SDS-PAGE and perform
quantification by phospho-imager analysis. But here is my problem - I see a
double band of MBP - one at 20 kDa and one at 14 kDa. It actually looks like the
MBP is degraded.
Has anyone experience something like that - can MBP be degraded during the
boiling of the sample before loading to the gel. Has anyone tried NOT to boil
the sample before loading the gel - or does that make any sense?!
Thank you in advance
Marianne Scheel Fjording
LEO Pharmaceutical Products