cells, fibroblasts, and different types of blood cells. Although in
minority, these other cell types could contribute with AMPK het-
erotrimers not present in the myofibers themselves. Little is known
about the regulation and substrate specificity of the different het-
erotrimeric complexes within the muscle cell. There could be dif-
ferences in upstream and downstream signaling, localization, and
metabolite sensitivity. Acknowledging that the analyses were not
specific to myofibers alone, but the whole mixed muscle tissue, we
set out more than 10 years ago to identify which of the 12 possible
heterotrimeric complexes is present in skeletal muscle and what the
stoichiometry of these might be [5–7]. To do this, we immunopre-
cipitated the different subunit isoforms and evaluated these for
coprecipitation of complex partners. This method requires strong
protein-protein interaction between the complex partners that have
to withstand the process of muscle tissue homogenization and the
immunoprecipitation procedure itself. The pitfall of this procedure
is false negatives that do not show up as complexes because the
protein-protein interaction is too weak to last throughout the
procedure. Another issue is the specificity of the antibodies used
for the precipitation. In theory, these could be more or less specific
toward certain complexes and may be blocked out by others if a
certain complex partner would hide the epitope. To avoid these
problems, we insured that the antibodies used were able to immu-
noprecipitate all of the specific protein they were targeted against—
leaving no fraction out of the equation. This procedure enabled
comparison as to how much a specific isoform coprecipitated the
others and vice versa. Putting these data together, it was possible to
account for the vast majority of the subunit isoforms and the
complexes they were part of.
The immunoprecipitation (IP) can be analyzed in two ways.
One way is to immunoprecipitate the different subunit isoforms
and load an equal amount of the IPs on the same gel and then blot
for one of the isoforms. This gives a direct comparison of how
much of the subunit isoform blotted for that is coprecipitated
with the others. Problems could be uneven antibody-antigen affin-
ity and hence washing away more of some isoforms than others in
the various IPs.
Analysis of the Post-IPs is a stronger method to compare
coprecipitation. By analyzing the same amount of sample protein
on Western blot for the Post-IP and the Pre-IP (~input), it is
possible to quantify how much of a specific isoform has been
removed by the IP procedure. This analysis is not dependent on
the washing steps of the IP, which makes it more consistent than
analyzing the IP per se. This way it is possible to control that an
antibody is able to immunoprecipitate all of the targeted protein.
However, since the samples are being analyzed and verified by
Western blot, there are limits to the outer extremes of the spec-
trum. On one end the analysis is restricted to the lowest detection204 Jesper B. Birk and Jørgen F. P. Wojtaszewski