Recently I tested my kinase activity by using phos-tag, and I need a control to confirm the kit is working. Do anybody know where can I by the cheap kinase and the substrate together?
For a protein kinase? I've had a lot of luck assaying activity with Promega's Peptag Non-radioactive assay kit which has control kinase to test against. It's essentially a gel shift assay based on a change in the peptide's isoelectric point after phosphorylation, and subsequent detection with the substrate's fluorescent tag. Very quick, very easy, and the results are clear to understand.
Thanks Larry, but I only have the phos tag kit, and I need use this kit to test my protein, so what I need is a positive control.
Can you provide better details for what it is you are looking for? There are vendors that sell protein kinases and substrates, but the info you have is too vague to make suggestions.
If you want to know whether your substrate is phosphorylated, can't you just run the mass spec of it?
My substrates are proteins, so I need the control is also a kinase, and the substrate should be proteins, for I don't know whether my protein has activity, my boss ask me to use phos tag gel to test it, however, I don't know whether the kit is working, so I need a control which can show me when the protein has been phosphorylated, it can be detected by this gel.
So what I need is a kinase whose activity has been confirmed and it's special substrate (should be protein)
Thanks
My experience with phos-tag acrylameide is that it's highly protein specific. Its common for two similarly sized phosphoproteins to have very different rates of migration through the gel. The best negative control for a Phos-tag acrylamide gel is a sample of your substrate that is not phosphorylated. Unphosphorylated substrate should run faster (farther) on the gel than phosphorylated substrate. If you see no retardation of the migration of your substrate protein band upon treatment with kinase than you are either not phosphorylating your substrate or the Phos-tag acrylamide is not sufficiently slowing the migration of your specific protein. Unfortunately, if you do see a phosphorylation-induced retardation in migration of a different substrate-protein that doesn't tell you whether or not your substrate-protein is being phosphorylated. In my opinion, the better positive control would be to use radiolabeled ATP as your phosphodonor and monitor where the radiolabeled band appears in the gel with film or a phosphoimager.
On a practical experimental note you have much more flexibility if you pour your own Phos-tag acrylamide gels than if you use the precast gels. You will likely have much better looking and more quantifyable gels if you spend some time optimizing Phos-tag, cation, acrylamide, and crosslinker concentrations than if you use the stock precast gels.
Thanks for Christopher, but my problem is for my protein is a new protein, the blast result shows it is a kinase, and according to the published work, the similar protein should phosphorylate two proteins, but we don't know our protein is as same as theirs. Now I run phos tag gel to test its activity, the results shown there are no different between the negative control (my substrate only) and the reacted systems, so there are two possibilities, one is my protein don't have activity, one is the phos tag gel did not work, so what I do is to find a positive control to test the phos-tag gel.
Jianbo, I understand your dilemma, but an unrelated kinase is not a very useful positive control. If you just want to see if Phos-tag acrylamide can separate a phosphorylated protein from an unphosphorylated protein you can use casein and alkaline phosphatase. Both reagents are cheap and come in solid form and they are what the Kinoshita group used used to validate the technology when they developed it.
Even if you find that your phos-tag gel does separate phosphorylated from unphosphorylated proteins it doesn't tell you whether your kinase is functional or not. Phos-tag just might not wok for your protein substrate, or only a very small fraction of your total substrate is phosphorylated. Since you don't have much information about the activity of your kinase your best bet is to monitor kinase activity directly, I would suggest using 32P-ATP as a nucleotide-substrate and seeing if you get any 32P transfer to your protein-substrate. With this method you can tell if even a small fraction of your protein-substrate is being phosphorylated.
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