AMPK Methods and Protocols

also be used, depending on protein recovery and other require-

ments. Recover cells by centrifugation at 7000gfor 30 min

at 4C, and freeze the pellets in liquid nitrogen.

4. Lyse cell pellets using a mortar and pestle under liquid nitro-
   gen, transfer powdered cells to 50 mL Falcon tubes, and resus-
   pend in lysis buffer. Typically 1 L of bacterial culture requires
   10 mL of lysis buffer. Clarify the lysate by centrifugation at
   100,000gfor 30 min at 4C and apply supernatant to an
   affinity-based chromatography column at a flow rate of 1 mL/
   min.


5. Wash the column in ten column volumes and carry out elution.
   GST-tagged proteins are eluted in buffer containing 20 mM
   glutathione at a flow rate of 1 mL/min, while His-tagged
   proteins are eluted using an imidazole gradient. Typically the
   gradient (20–500 mM imidazole) is performed at 1 mL/min
   over 30 min. On elution of protein (based on UVabsorbance),
   the gradient is stopped until absorbance at 280 nm has
   returned to baseline, and the gradient is then resumed.


6. Fractions containing the desired protein (usually detected by
   SDS-PAGE) are pooled, concentrated, and flash frozen in liq-
   uid nitrogen. AMPK protein is first dialyzed (41 L of dialysis
   buffer for 30 min) before being flash frozen.

3.3 Phosphorylation
of Bacterially
Expressed AMPK Using
CaMKK2

AMPK expressed in bacteria is not phosphorylated on Thr172 and

has a very low basal activity. This “naı ̈ve” unphosphorylated protein

can be used to study allosteric activation or promotion of phos-

phorylation. However, if the user requires AMPK protein phos-

phorylated on Thr172, then this will have to be carried out using

either LKB1 or CaMKK2. LKB1 is a heterotrimeric complex and

for full activity must be expressed in complex with STRAD and

MO25; for this reason CaMKK2 is easier to use. Pilot studies are

first performed to determine how much CaMKK2 is required to

phosphorylate a given amount of AMPK.

1. Mix 5μL of AMPK (0.1μg/μL) and 5μL of CaMKK2 (various
   amounts) and store on ice. Start the reaction at 15 s intervals by
   adding 10μL of unlabeled Mg.ATP, vortexing, and incubating
   at 30C for 10 min. Blanks lacking AMPK should be per-
   formed to ensure CaMKK2 is unable to phosphorylate the
   AMPK substrate peptide.


2. At 15 s intervals, remove 5μL from the first assay and add to
   20 μL of kinase assay mix (seeSubheading3.4) pre-dispensed
   into 1.5 mL microcentrifuge tubes, vortex, and incubate at
   30 C for 10 min.


3. Complete the kinase assays of this pilot as in Subheading3.4.

76 Fiona A. Fyffe et al.