- SDS-PAGE gel percentage must be optimized to ensure max-
imum separation between AMPK subunits and target proteins
of interest. The molecular weight of AMPK according to each
subunit is as follows:α(64 kDa),β(30 kDa), andγ(38 kDa)
subunits of AMPK. - It is recommended to use both positive and negative controls.
Negative controls can also be used to identify proteins that
contain autophosphorylation activity under the experimental
conditions. - The ratio of kinase to target protein in the kinase reaction
assay may be adjusted, but be aware that nonspecific phos-
phorylation is more likely to occur as the concentration of
AMPK and target substrates are increased. - As AMPK has autophosphorylation activity, reactions con-
taining AMPK alone can be used as a control [1]. However,
it may also be necessary to use reactions containing a known
substrate of AMPK such as acetyl-coA carboxylase (ACC) as a
positive control [9]. - Reaction incubation time may be adjusted; however, 1 h for
kinase reactions is usually sufficient to obtain positive
autoradiographs. - Gels that are not dried prior to developing the autoradio-
graph film may have some radiation shielded. If no auto-
radioactivity is detected, drying the gel may intensify the
signal. - The autoradiograph exposure time depends on the level of
radiant intensity of each sample. If no radiation is detected
with short exposure times, increasing the exposure time to
12 h or even several days may be beneficial [10].
Acknowledgments
This work was supported in part by NIH research grants R01
HL89940 and R01 HL108735 (JS) and Loma Linda University
GRASP 699349 (BG), GRASP 699330 (TM), and GRASP
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