I want to identify the relative quantification of all kinase proteins present in my 6 sample (sample is from rat tissue). Does anyone have any idea how can I perform this experiment? I am looking for some protocol through that I might pull down the respective kinases from all the samples.
Is it possible that I can relative quantify all the phosphoproteins present in the sample and look for only the kinases molecules?
Does this kind of experiment will give me the valid results for the kinase as I will be searching all the phosphoproteins?
Any kind of help or suggestions is welcome and appreciated.
This is a quantitative proteomics experiment. You will need to use isobaric labeling of the samples for the quantitative aspect. (https://en.wikipedia.org/wiki/Isobaric_labeling).
Thanks for the reply, but this not helpful I know I can do the relative quantification through iTRAQ or TMT or even Label-free quantification. But before doing that I want to separate only the kinase molecules (proteins) from all the samples so that I can compare them in each condition.
So, the question is how to isolate only the kinase molecules from the complex proteome?
One thing all kinases have in common is that they bind to ATP (although there are many non-kinases that also do that). Perhaps you could use an ATP affinity agarose (commercially available) to enrich for all the ATP binding proteins in an extract.
Thanks for the help, this is really something I can try. But I might get many other proteins too along with.
How to get rid of this?
Recently, I have learned about the kinomebeads. These beads contain 8 different type of affinity compounds attached specific to kinases. Do you have any idea related to this? or any other more specific molecules that can be used for the pulldown assay so to increase the kinase specificity.
I appreciate the help thank you.
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