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Mitochondrial kinase? - geting the wild side of putative kinase (Jan/08/2007 )

Hi, as anywody experience charaterisizing a putative mitochondrial kinse? We are interested in a S. cerevisiae gene harbouring a putative kinase domain and would like to find out about biochemical function and potential targets. I am having the hell of a time trying to purify it, as far as I know any kinase assay requires highly purified protein… I wonder… is there any way to point out putative kinase function without having the protein? We’ve got the null mutant, I was thinking what the hell, let’s get mitochondrial extracts and run 2D gels using phosphoprotein gel staining and compare wt versus mutant… does it sounds too crazy? wacko.gif wacko.gif

-Ganawa-

Some assays dont need highly purified protein for assay. U could try enzymatic assays with mitochondria prepared by subcellular fractionation.

Its easier said with the 2D gel than actually go through the whole procedure and trying to find differences between control and ur samples.

Look for other ways to purify ur protein, this might be easier.

-scolix-

Have you tagged the protein in some way? From what I know of yeast there are strains available that contain every protein with a tap tag. If you can get your hands on a strain with you protein tagged, then bob's your uncle. You can do an IP with the tap antibody and do a very standard in vitro kinase assay to test activity. You could always insert the tag yourself. This is actually quite easy, although I don't know how to do it!

I wouldn't go the 2D route. Phospho antibodies are rubbish. Plus, just because a protein would be more lowly phosphorylated in your mutant, doesn't mean your kinase is phosphorylating it.

-jaknight-

QUOTE (jaknight @ Jan 8 2007, 08:11 PM)
Have you tagged the protein in some way? From what I know of yeast there are strains available that contain every protein with a tap tag. If you can get your hands on a strain with you protein tagged, then bob's your uncle. You can do an IP with the tap antibody and do a very standard in vitro kinase assay to test activity. You could always insert the tag yourself. This is actually quite easy, although I don't know how to do it!

I wouldn't go the 2D route. Phospho antibodies are rubbish. Plus, just because a protein would be more lowly phosphorylated in your mutant, doesn't mean your kinase is phosphorylating it.


It came to my hands "proQ-diamond phosphoprotein gel stain" from Mol Probes, a “breakthrough technology that provides a method for selectively staining phosphoproteins in polyacrilamide gels” has anybody tried it?
I N-terminal His-tagged my protein using a Novagen vector, it turned out to be quite insoluble and eventually I improved it adding some sarcosyl 0.5%, I am still working on it but I am concerned about using a prokaryotic expression system for a mitochondrial protein. We didn’t took off the mitochondrial sequence because we are not pretty sure about it, the only info we have is on seq comparison (but we know for sure it is localized in the mitochondria)

-Ganawa-

QUOTE (scolix @ Jan 8 2007, 05:48 PM)
Some assays dont need highly purified protein for assay. U could try enzymatic assays with mitochondria prepared by subcellular fractionation.


Any kind of fractionation or proteomics is not a problem, but this kinase stuff is really new to my whole lab, honestly we are not familiarized with any kinase assay… could you please be more specific? I’d really appreciate it unsure.gif

-Ganawa-

I have sent a PM with a paper which describes an enzymatic assay for a mitochindrial kinase (Pank2). This should help you.

You could as well search for other assays on the net.

-scolix-

QUOTE (scolix @ Jan 9 2007, 06:18 AM)
I have sent a PM with a paper which describes an enzymatic assay for a mitochindrial kinase (Pank2). This should help you.

You could as well search for other assays on the net.



Hi Scolix. do you mind sendin that paper again. I'd appreciate it, as I am in a situation of developing a kinase assay for an unknown kinase.
So far I have bound a recombinant His-tagged substrate to NiNTA agarose, incubated with extracts (WCEs, cytosolic or mitochondrial), washed, and assessed kinase activitiy via 32P-ATP detection. No matter what I do, there is similar phosphorylation in vitro that is should be enhanced versus controls.

Thanks
David

-DOCDTT-

I have sent the paper. Check PM.

-scolix-