heterotrimeric complex. This has previously been shown for the
α 2 β 2 γ3 complex by correlating theγ3-associated activity to theγ3-
associatedα-Thr-172 phosphorylation [11]. In line with this, we
get strong correlations between our summed activities from mea-
surements of the activity of all heterotrimeric complexes in a set of
samples and the result from a phospho-AMPK Thr-172 Western
blot. This shows the robustness of the assay but also the importance
of dissecting the AMPK activity into different heterotrimeric com-
plexes. The activation of the different complexes differs greatly with
various interventions, and these differences are not always observ-
able in a total phospho-AMPK Thr-172 Western blot [11].
The assay described in the following is very similar to that
described by Davies SP. et al., in 1989 [3],and with immunopre-
cipitation by Vavvas D. et al., in 1997 [8]. It can be done either in
single Eppendorf tubes or in a medium throughput fashion using
96-well PCR-plates. The principle is the same as is the workflow,
but only the medium throughput fashion will be described here.
The duration of the assay (4–5 days) is not much shorter with the
medium throughput compared to the single-tube assay, but
approximately eight times as many samples can be run within the
same time frame. The medium throughput assay requires some
special equipment, which is not needed for the single-tube assay.
The Subheading3 is divided into three subsections, the first
describing the immunoprecipitation of the various heterotrimeric
complexes, the second the kinase reaction itself, and the last the
calculations for the specific activity of the samples.2 Materials
Prepare all solutions using ultrapure water (Milli-Q purified demi-
neralized water) and analytical grade reagents. Prepare and store all
reagents at room temperature (unless indicated otherwise).2.1 Immuno-
precipitation (IP)
- IP-buffer (seeNote 1): 20 mM Tris-base, pH 7.4, 50 mM
NaCl, 1% (v/v) Triton X-100, 50 mM NaF, 5 mM
Na-pyrophosphate, 500 μM phenylmethylsulfonyl fluoride,
2 mM dithiothreitol (DTT), 4μg/ml leupeptin, 50μg/ml
soybean trypsin inhibitor, 6 mM benzamidine, 250 mM
sucrose. This buffer is stored at 4C. - Protein A or G agarose (seeNote 2) washed thrice in IP-buffer
and used as a 50:50 slurry suspension. Stored at 4C. - Primary antibodies against desired AMPK subunit isoforms (see
Note 3). - 96-Well PCR-plates with V-shaped well bottoms and a volume
of 200μl per well.
Kinase Activity of Specific Heterotrimers 217