Immunoprecipitation wash + urea? - Isn't it too strong? (Oct/14/2008 )
Hello;
I've stumbled upon a co-immunoprecipitation protocol that uses a buffer with 1M urea in the final washing step (when the protein and the antibody are already bound at the beads). Isn't it, like, denaturing proteins and making them fall off the beads? Should I for the start make a less stringent buffer?
-Telomerase-
QUOTE (Telomerase @ Oct 14 2008, 05:39 AM)
Hello;
I've stumbled upon a co-immunoprecipitation protocol that uses a buffer with 1M urea in the final washing step (when the protein and the antibody are already bound at the beads). Isn't it, like, denaturing proteins and making them fall off the beads? Should I for the start make a less stringent buffer?
I've stumbled upon a co-immunoprecipitation protocol that uses a buffer with 1M urea in the final washing step (when the protein and the antibody are already bound at the beads). Isn't it, like, denaturing proteins and making them fall off the beads? Should I for the start make a less stringent buffer?
that is elution of antibody and protein off your beads. some people do that because they don't want to add SDS-PAGE buffer, so that they can use the IPed protein(s) for ELISA or other experiments.
what I do is that I use 0.1 M acetic acid in 0.5% Chaps. then neutralize with 0.15x volume of 1 M Tris, pH 8.
For positive control, I solubilize in 1% Triton x-100.
you can also see this thread of mine with a similar question. read mdfenko and dr.house's posts
Click here
-Curtis-
Thanks!
Actually the protocol means it's a washing step. Strangely stringent for me.
-Telomerase-