buffer for each sample. This minimizes the time the muscle is at
4 C where enzymatic processes slowly start up. Once the tissue
is lysed, the proteins and enzymes are extracted and mixed with
protease and phosphatase inhibitors, and then the time pres-
sure for having the sample at 4C is lowered.
- The Protein A or G agarose is usually supplied in a suspension
containing ethanol and has to be calibrated in the IP buffer.
When pipetting the agarose, use wide orifice pipette tips and
vortex or stir the suspension just before pipetting.
- The Pre-IP is the same as the input material to the IP procedure
and could be the raw lysate. Mixing the lysate with agarose
beads and antibody and incubating it overnight could however
make small changes in the appearance of protein bands on a
SDS-polyacrylamide gel (also proteins not precipitated). To
make the best comparison between Pre- and Post-IP, a control
IP with an unspecific/irrelevant antibody should be made, and
the Post-IP of this should be used as Pre-IP (input).
- The amount of antibody to be used in an IP depends on the
amount of lysate precipitated on and the affinity of the anti-
body, which must be tested. Usually 2μg of antibody is suffi-
cient for IP on 200μg of lysate. The concentration of antibody
varies between batches, and also to avoid pipetting very small
volumes, it is recommended to dilute the antibody in IP buffer
to a concentration of 0.2 mg/ml and then pipette 10μlto
each IP.
- The amount of lysate protein put into the IP depends on the
purpose of the IP. Searching for co-IP of AMPK heterotrimeric
complex partners, the IP and Post-IP must be used for several
Western blots detecting all seven subunit isoforms. The
amount of protein loaded on gels for these blots depends on
the antibodies used for the blots, but are in the range of
5–20μg, and some isoforms with different molecular weight
can even be analyzed on the same gel. Precipitating on 200μg
of lysate protein would give material enough for loading at least
five gels.
- The time and temperature for an IP can vary a lot depending on
antibody affinity, considerations for protein complex stability,
and workflow. Low temperature (4C) lengthens the IP pro-
cess, but maintains a higher stability for protease and phospha-
tase inhibitors as well as for protein interactions. Doing
consecutive IPs and measuring enzyme activity on each of the
IPs would challenge the number of hours in a working day if
the time for each IP was set to only a couple of hours. Our
experience is that the AMPK heterotrimeric complexes are very
stable and can endure at least three consecutive overnight IP
incubations at 4C.
Analysis of Heterotrimeric Complex Stoichiometry 211
