This may sound strange, but it is true.
I'm working on a bacterial histidine kinase, PhoR of B. subtilis. I
expressed it in E. coli, purified as a GST-fusion protein, while it's
still bound to the GST-agarose matrix, the protein was phosphorylated by
ATP which was subsequently washed off the matrix. The phosphorylated
protein was cleaved off the GST tag using thrombin. The cleaved protein
kinase was then left at room temp. for different time period and then
added to SDS sample buffer, run on the gel, quantified the radioactive
label for each time point to obtain a half-life curve.
interesting thing is: the halflife of the phosphorylated PhoR protein is
only about 15 min, using the GST-agarose pretreated with buffer. The
halflife of the same protein using GST-agarose heated in a boiling
waterbath for 10 min is more than 3 hours! The only difference between
these two experiments are the GST-agarose heat treatment.
I called Sigma to ask for hints, but the woman apparently didn't have
much idea of what she was talking about. She said that could not happen
because agarose would melt above 50c. :-))))) what else can I say?! I
think Sigma has the most useless tech-support that I have ever delt
with. ( not only from this time )
So I'm wondering if anybody ever had similar experience with respect to
GST-agarose. I would appreciate any response.
Actually, this does not bother me much more than the fact that Sigma
tech people know only agarose will melt. My experiments went fine.
But still, if there is phosphatase contamination, they should improve,
since I believe there are other autokinases are been studied using this
system.
Thanks a lot.
Lei
