I want to confirm my target protein can activate Rac1. I used Active Rac1 Pull-Down and Detection Kit" of Lifetechnologies. I over-expressed my target protein and followed the steps as indicated in the kit with 500ug of lysate but I could not detect the band of Rac1 activation. Only the positive control can observe the band.
What should I improve to observe the activate Rac1 band?
Thank you!
I have to admit, I have never used this kit. In case you are looking for an alternative technology to confirm binding and also to determine the binding affinity, you could consider MST (MicroScale Thermophoresis). If you express your protein as a fluorescent fusion protein, you even do not have to purify your protein, but you can just work with the lysate.
I attached a review for you proving more information about the MST technology in general and including an example of MST measurement in lysate.
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