I was used to follow the basic protocol for protein kinase assays incubating the sample with radioactive ATP and the purified kinase protein. Later I run the samples in an acrylamide gene and I dry it, but now I do not have a gel drier. Can I modify the protocol anyway without having to dry the gel?
I haven't tried it, but maybe you can blot the phosphorylated protein on a membrane and continue with the membrane instead of the dried gel.
Hi, I think you can try to seal your gel between two slides (or zipper bag). Then, set your gel and image plate (or film) in the cassette. After that, put the cassette at -80C. It should work. :)
As Franz mentioned,you can try it by blot the sample onto a membrane after SDS-PAGE and probe with anti-phospho-tyrosine(or serine,threonine)antibody that recognize phosphorylated tyrosine,serine or threonine residue regardless of surrounding sequence.Hope it will help.
I thought you transfer the proteins to nitrocellulose and do a Western. Or if your substrate is a protein, csn't you just remove the ATP from the protein by a spin column, ie Zebra buffer exchange column from Pierce, where the protein comes though and the ATP sticks, after spinning, and then count the flow through. You would probably have to do the gel first as Arhenius suggested. But for multiple assays, I don't know if a gel is necessary.
There are nitrocellulose filter binding assays that are used all the time. These too require no gels.
I never dried gel in protein kinase assay. Pack the gel using a plastic warp after the running and then directly expose the gel to the film in a -80 freezer is very convenient.
You can either dry your gel without a geldryer or use a scintallation counter. All you need to dry your gels is a large glas plate and appropriate cellophane sheets (they are not too expensive). One sheet on the bottom, one at the top, add support on the sides and clip into place with binder clips. For support you can order a geldrying frame or use wooden spatulas (we use ice cream sticks).
Alternatively, use a scintillation counter to measure your assay outcome (see Philip Cohen Nature Protocols paper for the method). You won't be able to see the size of your labelled substrate, but the method is more sensitive and gives you actual numbers to play with. It's super important that you use the correct controls here to make sure your activity is not due to background...
Depends on your kinase and substrate, really.
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