Problem in immunoprecipitation - (May/28/2004 )
I got a problem with my I.P. I'm trying to immunoprecipitate a FLAG tagged protein from a transgenic worm lysate (total lysate) using anti-FLAG Ab-ProteinG-sepharose. When I analyze equivalent amounts of total lysate before IP, bead extract, total lysate after IP through western blotting, almost all (at least 90%) the tagged protein bands were disappeared in lysate after IP, while strong in lysate before IP. However I couldn't see ovbious band either in the bead extract. My question is 'Where is the disappeared bands?'
Your IP worked but the target protein has been rendered insoluble by the procedure. Add a chaperone like DnaK (Sigma Chemical Co) to see the disappeared bands again.
Can you explain to me in a bit more detail? I'm actually trying to do a similar experiment with transgenic worms pulling down flag-tagged protein. One of my + controls is also GFP tagged: I can pull that down using GFP buit not Using FLAG!! I IP with Anti flag M@ from Sigma and Prot G beads and also detect with Anti FLAG M2. I was thinking about another Flag clone but my protein is C-terminally tagged with flag. I don't see it in total lysate nor in my IP
Here's the really funny part: I got a flag tagged protein lysate from cells from someone and I can detect that on the same blot??? So what is going wrong here? I (and my boss) are gonna hang myself!!!