[Protein-analysis] regarding immunoprecipitation

Ramachandran Balasubramanian via proteins%40net.bio.net (by lipid_ram At biochem.iisc.ernet.in)
Fri Nov 24 05:54:17 EST 2006


hello everyone,

I have been working on immunoprecipitation ie pull-down of my protein
using a monoclonal antibody and then I would use the pulled-down sample
for radiolabeled assays. But I could'nt get a clear picture of my
pull-down. I would be glad if anyone could help me. My procedure is,
I incubate my sample with antibody for overnight at 4 degree C, and then
add ptn. A agarose and incubate for about 12-16 hrs. Then I separate the
sup, the washes (with 1X PBS) and the pellet. But I could find activity in
all fractions ie sup, washes and pellet. I have also tried with
preclearing the sample with ptn. A agarose.

Can anyone suggest a good procedure for pull-down assays. or is my protein
interaction has some problem.

Thank you in advance,

sincerely

Ramachandran B
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