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Kinase assay problem

kinase assay MBP phospho-threonine

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#1 liflaf1

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Posted 19 January 2012 - 01:46 AM

Hi All,

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?

Thanks!

#2 fortunate

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Posted 25 January 2012 - 01:14 PM

try a different control protein other than BMP, and a different antibody. Did you add EDTA and EGTA to your buffer? ATP+SDS+substrate+divalent ions can cause chemical phosphorylation during sample boiling.

#3 liflaf1

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Posted 26 January 2012 - 07:37 AM

Thanks for your reply. When you ask if I added EDTA/EGTA, do you mean adding them to the stop solution? Because my stop solution was basically adding sample buffer (no EDTA or EGTA) and then simply boiling the sample. My kinase buffer, however, contained low EDTA/EGTA levels: 1mM EDTA and 0.4mM EGTA.

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!




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