Posted 19 January 2012 - 01:46 AM
I'm trying to develop a non-radioactive kinase assay using MBP as a substrate. The plan is to perform the kinase reaction (using 50uM cold ATP - no hot ATP at all), then add sample buffer and run on SDS-PAGE, followed by western blot using anti-phospho-threonine antibodies. My problem is that I get bands that correspond to the MBP MW) even when no kinase is present. The signal does not increase with increasing concentrations of my kinase. I should say that the kinase is commercial, and should be active. Here are the conditions:
Rxn vol: 25ul
ATP (stock powder is dissolved in pH 7.2 buffer) final: 50uM
MBP (sigma cat# M1891): 5000ng/reaction
Antibody: anti-P-Thr from Cell Signalling cat# 9381
What can I do to reduce the initial ('no enzyme') MBP signal, and to generally improve the assay?
Posted 25 January 2012 - 01:14 PM
Posted 26 January 2012 - 07:37 AM
Also, I noticed there is a dephosphorylated MBP (by Millipore for example). Is this one better than the Sigma MBP, which is not treated?
Thanks in advance!