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?'
-yjokim-
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.
-phdconsult-
QUOTE (phdconsult @ May 30 2004, 08:19 PM)
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!!!
-Rintelman-