Is there anyone who can provide information about using anti-V5 tag antibody for immuno-precipitation?

15 August 2014 0 9K Report

I need some advise regarding an immuno-precipitation (IP) experiment I have been working on. I am trying to precipitate V5-tagged proteins from cell lysates using anti-V5 antibody (Thermo Scientific catalogue #PIMA5-15253). As control, I have transduced cells with lac z gene so that the cells make b-galactosidase protein containing the V5-tag. When I perform the IP with the anti-V5 antibody, I am expecting my protein of interest to be precipitated from the cell lysate and PP2A to co-precipitate with the protein. I do not expect PP2A to co-precipitate with b-galactosidase from the cells transduced with lac z. However, for some reason, PP2A is co-precipitating with b-galactosidase. I have tried using different beads and different antibodies to perform the IP experiment, the results remain the same. Has this happened to anyone? I need some advise as to what I can do now. Is there a way to purify the V5-tagged proteins and then cleave the tag?

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