I've been trying to optimise some IPs lately and having some problems:
1.) In my negative controls (where I don't attach an antibody to my beads, and just incubate with whole cell lysate) I'm still getting a signal after WB in the 50 and 25 kDa range
2.) I'm struggling to precipitate my protein of interest (no band at the expected size)
Originally, I was able to get the IPs to work fairly well, but now I'm struggling to get anything useful at all I'm using protein A sepharose from sigma, and my antibodies have worked for IP before.
Could it be that my beads have somehow become contaminated? They're kept in 20% ethanol in buffer at 4 degC(at sigma's recommendation), and I would have expected contamination to leave a big smear rather than distinct antibody-type bands. I'm very careful with changing tips etc, so I doubt that cross-contamination is my problem.
Is protein A known to break down readily? Could this be the problem?
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