AMPK Methods and Protocols

6. Okadaic acid: 10 μg/mL okadaic acid in DMSO. Store at
   
   20 C.


7. Recombinant His-AXIN (full length), expressed inE.coli.


8. Recombinant GST-ACC1 (aa 34–100), expressed inE.coli.


9. Thermomixer equipped with 1.5-mL reaction vessels.

2.4 Measurement
of AMP, ADP, and ATP

The reagents listed below should be of HPLC or higher grade.

Methanol and chloroform should be precooled to 4C prior to use.

1. Mannitol solution: 5% (w/v) mannitol in H 2 O.


2. Methanol.


3. Chloroform.


4. Ammonium acetate: 50 mM ammonium acetate, pH 8.5, in
   H 2 O.


5. Electrolyte containing phosphate buffer (seeNote 15), stored
   at 4C.


6. Sheath liquid: methanol/water (50% v/v) containing 0.1μM
   2,2-difluoroethoxy phosphazene, stored at 4C.


7. Internal standard 1 (IS1): 50μML-methionine sulfone, 50μM
   D-camphor-10-sulfonic acid in ultrapure water. Store at 4C.


8. Internal standard 2 (IS2): 50μM 3-aminopyrrolidine dihy-
   drochloride, 50μM N,N-diethyl-2-phenylacetamide, 50μM
   trimesic acid, 50μM 2-naphthol-3,6-disulfonic acid disodium
   salt in methanol. Store at 4C.


9. Ultrafree-MC-PLHCC centrifugal filters.


10. Freeze Dry System: Refrigerated CentriVap Vacuum
    Concentrators.


11. Capillary electrophoresis equipped with a pump and a splitter
    (1:100).


12. Mass spectrometry.


13. Fused-silica capillary (i.d. 50μm80 cm).


14. Injection vial.


15. Aluminum clamp (seeNote 16). The clamp should be prefro-
    zen in liquid nitrogen prior to use.

3 Methods

3.1 Analysis of AMPK
Activation in Low
Glucose (See Notes 17
and 18)

All media should be preheated at a 37C water bath prior to use.

Glucose Starvation and Determination of p-AMPKα-Thr-172

in MEFs

1. MEFs are cultured to 70–80% confluence (seeNote 19)in
   6-well dishes containing 2 mL of complete DMEM medium
   per well in a 5% CO 2 incubator.

398 Chen-Song Zhang et al.