Hello, I'm curious about the in vitro experimental method used to assess whether or not some kinase enzymes directly phosphorylate other kinase receptors, such as EGFR. Which experimental method is appropriate for this type of research? If you have a suggestion, please share it with me.
Hi, an in vitro kinase assay would be a good choice. This method would allow you to determine whether or not a particular kinase can phosphorylate a particular substrate, without having to worry about the complex environment of a living cell.
Depending on the tools you have at your disposal. I agree that the best starting point is in vitro assay. If your kinase is not constitutively expressed, you need to transfect it into the cell. In the presence of control (inhibitor of your kinase or transfection of kinase-dead plasmid), you can then blot for your phosphorylated proteins to see whether phosphorylation occurs or not. If you want to go a step further to show direct interaction, you can do Co-IP
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