an assay for a putative Michondrial kinase

2 posts / 0 new
Last post
GANAWA
GANAWA's picture
an assay for a putative Michondrial kinase

Very recently a S. cerevisiae gene of unknown function came into our attention because the product is associated to a mitochondrial metabolic pathway we are interested in, and by running sequence homology we suspect is a Kinase. It has some homologies with kinases C. Homologs in human, mouse, etc, havent been characterised either. We cloned it, we are purifying the protein (his-tag) and would like to know wheter is a kinase or not. How? Any good advise? Of course we have no idea what the substrate might be. I have been looking all this kits for kinase monitoring activity, but of course a substrate is required

Larkia
Larkia's picture
GANAWA wrote:Very recently a

GANAWA wrote:

Very recently a S. cerevisiae gene of unknown function came into our attention because the product is associated to a mitochondrial metabolic pathway we are interested in, and by running sequence homology we suspect is a Kinase. It has some homologies with kinases C. Homologs in human, mouse, etc, havent been characterised either. We cloned it, we are purifying the protein (his-tag) and would like to know wheter is a kinase or not. How? Any good advise? Of course we have no idea what the substrate might be. I have been looking all this kits for kinase monitoring activity, but of course a substrate is required

I know of at least 2 different providers of kinase substrate-screening kits you could use to find a substrate if your protein is a kinase. Cell Signalling has an antibody-based peptide screening kit, and Molecular Devices has kits based on their IMAP technology.

Is your protein more similar to Tyr kinases or Ser/Thr kinases? Molecular devices has separate kits for the 2 varieties, and the Cell Signalling kit primarily has Ser/Thr peptides.

However, if your protein is most similar to Tyr kinases, you may want to try a simple poly glu-tyr (4:1) substrate (as an ELISA or P32 assay) to see if you get activity with that, before you spend ~$3000 on a kit to look for a substrate. Unfortunately, Ser/Thr kinases are more substrate-specific.

One word of caution, though: Before you do a substrate screen, make sure you have a relatively pure protein. I've had some trouble with contaminant kinases in his-tag purified protein preparations. We purchased two his-tag purified kinases (~60% pure) from a major supplier. We did a substrate screen, found a good substrate, and even developed an assay, but when we reviewed our data, we realized that the substrate was a Ser/Thr substrate, and the enzymes (IGF-1R and JAK3) were Tyr kinases! Additionally, both enzymes had identical inhibitor profiles. Thus, we came to the conclusion that neither enzyme was actually responsible for the kinase activity, but that both enzyme preparations contained a contaminant Ser/Thr kinase which was responsible for the kinase activity.