limit of Western blotting. If nearly all of a subunit isoform is
coprecipitated, then the little remaining protein in the Post-IP
might be below the detection limit on the Western blot making it
look like all of the protein was coprecipitated. On the other end of
the scale, i.e., coprecipitating only a small part of a subunit isoform
fraction, it might be hard to determine if the Post-IP band is indeed
weaker than the input (Pre-IP) band. In this case, however, the IP
itself can help to verify the coprecipitation, because it would show a
faint band of the coprecipitated protein.
The Subheading2 is divided into two subsections: one describ-
ing the homogenization of muscle tissue and the other the immu-
noprecipitation procedure. In theory, the IP procedure could work
on any lysates produced by homogenization in different kinds of
homogenization buffers. However, a short description is given to
the homogenization protocol because we have experienced that
other buffers (e.g., RIPA buffer (Sigma R0278) and Rac1 lysis
buffer (Cytoskeleton Inc. BK126)) induce problems if the IP is
followed by an enzyme activity assay (described in Chapter 14).
Due to high salt, but still mild detergent (Nonidet P-40), the
homogenization buffer we use is relatively strong and gives a high
protein yield in the lysate. This gives a higher extraction yield of
AMPK from the muscle tissue and lower chance of losing poten-
tially important fractions in the pellet when the homogenate is
centrifuged to get the lysate. The disadvantage could be disruption
of certain AMPK complexes, though comparing with milder
homogenization buffers this is not our experience. The homogeni-
zation buffer is possibly not strong enough to lyse the cell nuclei,
and thus our method does not detect AMPK complexes in this
compartment, unless mechanically disrupted by the tissue lysing.
Adding ionic detergents (e.g., sodium deoxycholate or SDS) would
solve this, but at the same time, most likely disrupt the AMPK
heterotrimeric complexes making co-IP impossible.
We do not go into details describing the total protein determi-
nation procedure, because this is considered as standard and will be
done equally on all samples involved in the subsequent IP proce-
dure. The main requirement for the assay for determination of the
protein concentration is that it is compatible with the ingredients in
the homogenization buffer, which is the case for the bicinchoninic
acid (BCA) assay.
The same applies for the SDS-PAGE and Western blot analysis
following the IP procedure, which should not change the outcome
of the IP procedure. Certainly, it is important that antibodies are
appropriately verified for specificity and the blots are tested for
signal linearity in relation to the amount of protein loaded on
the gel.
Analysis of Heterotrimeric Complex Stoichiometry 205