tearing my hair out over IPs
jlogan at genetics.utah.edu
Wed Apr 10 20:09:03 EST 1996
**this message is long. if you're in a hurry, save some precious
time and skip to the next post**
I am trying to use co-immunoprecipitation to confirm an interaction
between my favorite mammalian protein and a putative binding partner.
My results are encouraging but not quite clean, and I'm tearing my
hair out trying to get data that I actually believe. Here's the
Both of the proteins I'm working with have leucine zipper domains, and
it is possible, but not certain, that they would interact via
coiled-coil oligomerization. Each is expressed at low levels in
immortalized, cultured human (and other mammalian) cells. In fact,
expression of the candidate binding partner is virtually undetectable
at both the mRNA and protein levels in most cell lines we've examined.
Therefore, while I have antibodies to both that work well for
immunoprecipitation, getting enough of each protein out of cells to
detect an interaction is next to impossible. As a result, I've had to
"supplement" my "in vivo" experiments using the following approaches.
What I've tried: transfecting/overexpressing the candidate binding
partner in cells that do express my protein, then co-IP with an
antibody against my protein. The two proteins co-IP nicely, but the
candidate also precipitates with the pellet -- albeit at diminished
levels -- in control IPs using a nonspecific IgG. I've seen the same
result when I've tried to co-IP the two proteins translated in vitro
from retic lysate and in other variations on the same theme.
Background! The bane of my existence! I need help in getting rid of
Approaches/reagents that have failed:
1) Both protein A sepharose and protein G agarose give the same
results, even after blocking in 1% BSA.
2) Preclearing the lysate with protein A/G for 1 hour at 4° C doesn't
3) Lysis/wash buffers with higher salt (up to 250 mM KCl) and
detergent (0.1% NP-40, 0.1% Triton X-100, 0.1% SDS) don't make any
difference, but neither do they disrupt immunoprecipitation.
4) Even "high stringency" washes in RIPA buffer (1% deoxycholic acid,
0.1% SDS, 0.1% Triton X-100) do not disrupt precipitation of the
candidate binding partner.
5) My standard lysis/wash buffer is PBS + 0.1% NP-40, with the above
goodies thrown in.
I've basically banged on my co-IPs with a large hammer, but the
background won't budge.
Should I just drop-kick the whole idea? Is there anything I've missed?
(Besides doing the reciprocal co-IP, using antibody against the
candidate partner itself. I'm working on that.) I invite anyone to
point out the obvious to me or to clue me in on their foolproof
protocol. Please, before I go bald, help me out!
I'm sorry this message was so verbose, but I wanted to include the
details so that kind souls wouldn't waste their time suggesting things
I've tried. Also, any other suggestions regarding how one might go
about proving such an interaction would be welcome!
Thanks in advance,
**Really, I'm only in this for the money and prestige**
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