purified AMPK can be eluted from anti-FLAG M2 affinity
agarose gel using FLAG elution buffer instead of glutathione
agarose elution buffer. Note that CaMKK2 phosphorylation
cannot be performed on anti-FLAG M2 affinity agarose gel
because FLAG peptide present in CaMKK2 preparations gen-
erated by our method will result in undesirable AMPK elution
during the phosphorylation step.- Both β1 (30.4 kDa) and γ3 (54.3 kDa) subunits display
reduced electrophoretic mobility on SDS-PAGE, relative to
expected mobility based on molecular mass. - A second elution step can be performed. The second elution
typically possesses half the protein concentration of the first
with similar volume. - Yields can vary significantly between preparations, being influ-
enced mainly by cell viability, transfection efficiency, and the
recombinant AMPK complex being expressed. Typically, we
achieve yields of 1–2μg purified AMPK per 10 cm dish. - Nonreducing lysis buffer (devoid of DTT or reducing agent
equivalents) is necessary to avoid dissociation of the heavy and
light chains in the immobilized M2 antibody present on anti-
FLAG M2 affinity agarose gel. This consideration is important
for maximal yield.
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Production in Mammalian Cells 169