I have CaM bound sepharose beads. I also have a Purified protein 'X' and one of my cell line over expresses protein 'Y'. I need help with the experiment planning to look for these protein interaction in presence or absence of a drug that I am going to test. Normally CaM binds to protein X and hence we should see more protein Y. when drug is added we should see less Y. So i was wondering if an experiment could be planned like as follows: incubate together CaM beads and protein X. Incubate protein Y cell lysates into the mixture for 2 hrs along with drug or control and then elute, blot with Ab for protein X and Y look for the intensity differences. Thanks for the help in advance!!
Does the protein X bind CaM and protein Y at the same time? Because if not, this set-up will not work.
Maybe you can use several concentrations of your compound to see some dose-dependence.
Hi there,
As a first rough experiment, I would run 4 samples :
beads +X alone (to check X binding in absence of Y)
beads +X first then add lysate (to see if Y is able to binf to X/beads complex)
beads + X and lysate at the same time or premixed (to check if X/Y interaction is a prerequisite to interaction with beads)
beads + lysate alone (to check possible non specific Y binding to the beads in absence of X)
Test the effect of drug on the 4 samples.
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