- Compare the Post-IPs to the Pre-IP to see how much of each
specific subunit isoform is missing in the Post-IPs after the
various IPs. - Do not quantitatively compare signal intensities between the
IPs and the Post- or the Pre-IPs (seeNote 14).
4 Notes
- The IP buffer could be the same as the homogenization buffer
or even just phosphate-buffered saline (PBS). Our experience is
that analyzing enzyme activity after the IP (seeChapter 14)
gives higher specific activity with the milder IP buffer com-
pared to the homogenization buffer for reasons unknown.
Advantages of the IP buffer compared to PBS is the detergent
in the IP buffer which eliminates the surface tension and makes
the solution mix better in very small tubes during the end-over-
end incubation. Also the addition of extra protease and phos-
phatase inhibitors during the IP incubation is beneficial. - The selection of either Protein A or G agarose is dependent on
the species and subgroup of the antibody used in the IP. Most
antibodies work best with Protein G, but this should be verified
before start. As standard we use Protein G agarose, Fast Flow
from Merck (cat. #16-266). - It is paramount that the antibodies used for immunoprecipitat-
ing each subunit isoform are able to IP all of the particular
subunit from the lysate. In case a specific subunit isoform
cannot be precipitated (near) quantitatively, there is no way of
knowing if the remaining fraction is part of a special hetero-
trimeric complex or if it is a complex fraction that is posttran-
slationally modified by signaling that could be important for
the distribution and regulation of AMPK. Over the years, we
have used many different sources of antibodies as some have
run out of stock. All have been tested thoroughly before use
and during our search; many have been rejected for precipitat-
ing partially or not at all. At the time of writing, we have still
not been able to find suitable antibodies for IP of the AMPKγ 1
andγ2 isoforms. - If homogenizing a large portion of muscle (more than ~12 mg
freeze-dried or 50 mg of wet weight muscle), it is not advisable
to add more than 800μl of buffer to the 2 ml tube when tissue-
lysing, because too much fluid in the tube will slow down the
movements of the steel ball. Add the remaining buffer after
tissue-lysing before the samples are set to rotate for 1 h
(step 9). To speed up the process, add the same amount of
buffer to all samples, tissue-lyse, and then fill up the remaining
210 Jesper B. Birk and Jørgen F. P. Wojtaszewski