After you got some candidates that may interact with your protein of interests by either Y2H screening or immunoprecipitation+ LC MS, what would you do next?
validate interaction?
check mutants of those candidates?
what else would you suggest?
Thanks!
Hi there,
If you actually know the biological role of the complex, try to make a link between the interaction and the biological activity of the protein complex.
Thanks. From mutant analysis of the gene , we know its biological function. But we have no idea about its molecular function. So we tried to identify interacting proteins. Now we have a list- some of known proteins are there, and some of unknown proteins as well. It is not easy for us obtain/examine all mutants of those candidate genes in our plant system. Besides, genetic approach by examining mutants, any methods can narrow down the real interacting candidates? We used to BiFC to validate their interaction, most of them are positive. Thanks!
If you express and purify the proteins, you can measure the strength of the interaction (Kd) using biophysical techniques, such as surface plasmon resonance. If you are very ambitious, you can try to obtain a x-ray crystal structure of the complex, or use cryo-EM to get structural information.
For validation of interaction you can knock out the protein to see the function in up and/or downstream regulation. GFP tag your protein of interest (mutant copy and WT) and do cell cycle arrest. If these proteins are activated by kinases, you can perform a kinase array or do mutagenesis to elucidate which functional domains are needed for activation or degradation.
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