AMPK Methods and Protocols

(weigh 250 g glycerol, 25 g glucose, 100 gα-lactose, gently

heated and dissolved in 730 mL deionized water and auto-

claved at 110C for 10 min), 50 mL of 20NPS (weigh

66 g NH 4 SO 4 , 136 g KH 2 PO 4 , 142 g Na 2 HPO 4 gently heated

and dissolved in 900 mL deionized water and autoclaved at

121 C for 15 min).

7. Isopropylβ-D-1-thiogalactopyranoside (IPTG): 1 M IPTG.


8. Bacterial culture flasks.


9. Centrifuge and centrifuge pots.


10. Pestle, mortar, and liquid nitrogen.


11. Lysis buffer for AMPK: 50 mM Tris–HCl, pH 8.0, 500 mM
    NaCl, 20 mM imidazole, with EDTA-free protease inhibitor
    tablets dissolved according to manufacturers’ instructions.


12. Elution buffer for AMPK: 50 mM Tris–HCl, pH 8.0, 500 mM
    NaCl, 500 mM imidazole.


13. Dialysis buffer for unphosphorylated AMPK: 50 mM Na
    HEPES, pH 8.0, 200 mM NaCl.


14. Bacterial plasmid expressing CaMKK2 (e.g., [20].)


15. Lysis buffer for CaMKK2: 50 mM Tris–HCl, 500 mM NaCl,
    1 mM DTT, 1 mM EGTA, 1 mM EDTA, with EDTA-free
    protease inhibitor tablets dissolved according to manufac-
    turers’ instructions.


16. Elution buffer for CaMKK2: 50 mM Na HEPES, pH 8.0,
    200 mM NaCl, 20 mM glutathione.


17. Protein purification system (e.g., GE Healthcare A ̈KTA).


18. HisTrap FF and GSTrap FF columns.

2.3 Phosphorylation
of Bacterially
Expressed AMPK at
Thr172 Using CaMKK2

1. Gel filtration equilibration buffer: 50 mM Na HEPES, pH 8.0,
   200 mM NaCl.


2. Dialysis buffer for phosphorylated, active AMPK: 50 mM Na
   HEPES, pH 8.0, 200 mM NaCl, 50% (v/v) glycerol.


3. Protein purification system (e.g., GE Healthcare A ̈KTA).


4. GSTrap FF and HiLoad 16/600 Superdex 200 pg columns.


5. Dialysis tubing or cassettes.


6. Unlabeled Mg.ATP: 10 mM MgCl 2 , 400μM unlabeled ATP.

2.4 In-Solution
Kinase Assay

1. Phosphocellulose (P81) paper cut into 1 cm squares, num-
   bered with a hard pencil (if one corner is folded over, this
   makes the squares easier to pick up using forceps).


2. Kinase assay buffer: 50 mM Na HEPES, pH 7.4, 150 mM
   NaCl, 1 mM DTT, 0.02% (v/v) Brij-35. Store at room tem-
   perature, but do not add Brij-35 and DTT until immediately
   prior to use; otherwise, store in aliquots at
   20 C.

72 Fiona A. Fyffe et al.