AMPK Methods and Protocols

6. CaMKK2 phosphorylation buffer: 50 mM Tris–HCl, pH 7.4,
   150 mM NaCl, 1 mM DTT, 10% (v/v) glycerol, 2 mM MgCl 2 ,
   200 μM ATP.
   7.λ-phosphatase.
   8.λ-phosphatase dephosphorylation buffer: 50 mM Tris–HCl,
   pH 7.4, 150 mM NaCl, 1 mM DTT, 10% (v/v) glycerol,
   2 mM MnCl 2.

3 Methods

3.1 Expression of
Recombinant AMPK

1. Maintain mammalian cells (COS7 or HEK293) in sterile 10 cm
   tissue culture dishes in DMEMþ10% FBS, supplemented with
   penicillin and streptomycin. Incubate at 37C and 5% CO 2 /
   humidified air (seeNote 9).


2. 24 h prior to transfection, aspirate media, and wash cells in 8 ml
   of PBS at 37C. Aspirate PBS and detach cells by the addition
   of 5 ml trypsin/EDTA, followed by incubation at 37C. Cell
   detachment can be aided by gently agitating plates by hand.


3. Seed 10 cm dishes at 30–40% confluency (1.0–1.3 106 cells
   per dish), and incubate in DMEMþ10% FBS overnight at
   37 C and 5% CO 2 /humidified air.


4. For DNA transfections prepare DNA dilutions. First, prewarm
   DMEM and DNA expression constructs for each AMPKα,β,
   andγsubunits, to room temperature. For each 10 cm dish
   transfection, add 1μg each ofαandγDNA construct, and
   0.5–1.5μgofβDNA construct (seeNote 10), to 100μlof
   DMEM in a sterile 1.5 ml Eppendorf tube, and mix gently by
   flicking the tube or vortexing. DNA dilutions for multiple plate
   transfections can be prepared in the same tube. To prepare
   transfection complexes, add 9μl of prewarmed transfection
   reagent per 10 cm dish to DNA dilutions. Mix and incubate
   for 20 min (seeNote 11).


5. Add transfection reagent/DNA mixture dropwise to each
   10 cm dish. Tilt dishes by hand to mix, and return to incubator
   at 37C and 5% CO 2 /humidified air.


6. After 48 h, replace medium in transfected plates with fresh,
   DMEMþ10% FBS at 37C, and return to incubator at 37C
   and 5% CO 2 /humidified air.


7. After 2 h, remove dishes from incubator and place on ice at a
   slight angle. We recommend that the following steps proceed
   directly to avoid unnecessarily stressing cells leading to unde-
   sirable AMPK activation.


8. Aspirate media and apply 5 ml of ice cold PBS to the top edge
   of the dish.

Production in Mammalian Cells 163