-
Principle:
Co-
Immunoprecipitation (Co-IP) was developed from the
immunoprecipitation
technique with
which Co-IP shares the fundamental principle of
the specific
antigen-antiody reaction.
Co-IP helps determine whether two proteins
interact
or not in physiological
conditions in
vitro
.
Graphically, the Co-IP principle is as
described in the right hand side
picture.
The known protein
(antigen) is termed the bait protein, and the
protein it
interacts with is called the
prey protein. The standard Co-IP protocol is the
same as that described for IP, and
actually any system designed for IP should
also work for Co-IP.
After that cells are completely lysed
under non-denaturing conditions, proteins
that bound together are kept. Therefore
if you use anti-X to precipitate protein
X through Co-IP, then you can get other
proteins that interact with protein X
in
situ
.
Co-IP is
applied to test whether two known proteins bind
each other in cells,
or to find a new
protein that interacts with a known
protein.
Advantages:
1.
Proteins that
interact in a typical Co-IP are post-
translationally modified
and
conformationally natural.
2.
In Co-IP
proteins interact in a non-denaturing condition
which is almost
physiological.
Disadvantages:
1.
The signals of
low-affinity of protein interactions might not be
detected.
2.
3.
There might be
a third protein in certain protein-protein
interaction.
To choose an
appropriate antibody, the target protein needs to
be
properly predicted. Or there would
not be a positive result in Co-IP.
Reagents and
buffers:
?
?
PBS
RIPA (RadioImmunoPrecipitation Assay)
Lysis buffer:
Tris-HCl: 50 mM, pH
7.4
Nonidet P-40 (NP-40):
1%
Deoxycholate
Na
：
0.25%
NaCl: 120 mM
EDTA: 1 mM
*PMSF:
1 mM
*Leupeptin 1
μg/ml