from Promega; however alternative transfection reagents from
other suppliers can be used and their use optimized empirically.- We observe negligible protease activity during cell lysis using
cOmplete™ mini protease inhibitor cocktail tablets from
Sigma-Aldrich. These tablets provide a convenient means to
ensure consistency between different lysis buffer preparations.
Alternative protease inhibitor cocktails from other suppliers
may be used. - Glutathione agarose elution buffer can be prepared from glu-
tathione wash buffer by addition of 20 mM glutathione. If this
method is used, it is critical to return the pH of the elution
buffer to 7.4 with a few drops of 10 M NaOH prior to use. It is
not uncommon for inexperienced operators to disregard this
step. 20 mM glutathione will acidify the wash buffer to
~pH 3.2. Elution buffer at ~pH 3.2 is an ineffective eluent
and prevents subsequent attempts to recover AMPK using
elution buffer at correct pH 7.4. The preparation will have to
be discarded. - We find maximal yields are achieved using anti-FLAG M2
affinity agarose gel from Sigma-Aldrich. This affinity resin con-
tains anti-FLAG M2 monoclonal antibody covalently attached
to cross-linked 4% agarose beads, thereby reducing antibody
leaching during the prolonged incubation steps. - FLAG synthetic peptide (amino acid sequence DYKDDDDK)
is commercially available from a number of suppliers but can be
expensive in the event of large-scale preparations. We have
found custom peptide synthesis from any number of indepen-
dent services (e.g., GL Biochem (Shanghai)) to be an effective
and more economical source. - For master cell cultures, maintain confluency between 10% and
90%. Over-confluent cells become resistant to liposomal trans-
fection, resulting in reduced efficiency and lower protein yields.
Discard cell cultures at passage 40; transfection efficiency is also
diminished using over-passaged cells. - As outlined inNote 3, AMPK yield is dictated byβ-subunit
expression. If theβ-subunit is untagged, or myc-tagged, we
typically add 1.5μg DNA per 10 cm dish. Conversely, we
reduce to 0.5μg if theβ-subunit is being expressed as a
FLAG fusion. - The 20 min incubation should be strictly observed. Longer
incubation times result in drastically reduced protein yields. - The method describes purification via the GST-tag on
α-subunits but can be adapted accordingly for other affinity
tags. For FLAG, myc or HA purification, prepare lysis, wash
and elution buffers in the absence of DTT. If required, FLAG-
168 Jonathan S. Oakhill et al.