AMPK Methods and Protocols

Weigh 9.1 mg of AMP monohydrate and transfer to the glass

beaker container. Mix and adjust pH to 7.4 with Tris–HCl

powder. Make up to 25 ml with mQ water. Aliquot (200μl)

in 0.5 ml tubes and store at 80 C.

The determination of the exact final adenine nucleotide stan-

dard concentrations can be performed by UV spectrophotometry

using a quartz cuvette and the following formula: [ATP/ADP/

AMP]inμM¼optical density at 259 nm/15.4 (cm^1 .10^3 M^1 ).

3 Methods

Carry out all procedures at room temperature unless otherwise

specified.

3.1 Extraction
of Adenine Nucleotides
from Cultured Cells

1. Rinse cell monolayers (e.g., one million hepatocytes plated in a
   collagen-coated 4-cm-diameter Petri dish) once with
   ice-cold PBS.


2. Scrape the cells in 250μl of ice-cold perchloric acid/EDTA
   with a rubber policeman (seeNote 2).


3. Transfer the cell extracts containing precipitated proteins to
   1.5 ml tubes, vortex briefly once, and keep on ice for ~30 min.


4. Spin down precipitated proteins at 8000gfor 2 min at 4C.


5. Transfer 200μl of the supernatant fractions into a new 1.5 ml
   tube (on ice) and adjust pH to 6.5–7 with ~130μl of KOH/-
   MOPS. pH should be checked after brief vortexing using
   pH-indicator strips. pH>7 should be avoided.


6. Clarify lysate by centrifugation at 8000gfor 2 min at 4C.


7. Transfer 200μl of the (neutralized) supernatant fractions into a
   new 1.5 ml tube (on ice) and store at 80 C until determina-
   tion of nucleotide concentrations. Neutralization of the sam-
   ples before storage at 80 C is recommended in order to
   minimize adenine nucleotide degradation during freeze-thaw
   cycle.

3.2 Extraction
of Adenine Nucleotides
from Frozen Tissues

1. Lyse a piece of frozen tissue (e.g., 50 mg liver; kept at 80 C)
   in 500μl of ice-cold perchloric acid/EDTA using a Polytron
   homogenizer (three times 10 s).


2. Transfer immediately tissue lysate containing precipitated pro-
   teins into a new 1.5 ml tube and keep on ice for ~30 min.


3. Spin down precipitated proteins at 8000gfor 2 min at 4C.


4. Neutralize the supernatant fractions as described in Subhead-
   ing 3.1,steps 5– 7 and store at 80 C until determination of
   nucleotide concentrations.

232 Noemı ́Garcı ́a-Tardo ́n and Bruno Guigas