(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).
- Isopropylβ-D-1-thiogalactopyranoside (IPTG): 1 M IPTG.
- Bacterial culture flasks.
- Centrifuge and centrifuge pots.
- Pestle, mortar, and liquid nitrogen.
- 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. - Elution buffer for AMPK: 50 mM Tris–HCl, pH 8.0, 500 mM
NaCl, 500 mM imidazole. - Dialysis buffer for unphosphorylated AMPK: 50 mM Na
HEPES, pH 8.0, 200 mM NaCl. - Bacterial plasmid expressing CaMKK2 (e.g., [20].)
- 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. - Elution buffer for CaMKK2: 50 mM Na HEPES, pH 8.0,
200 mM NaCl, 20 mM glutathione. - Protein purification system (e.g., GE Healthcare A ̈KTA).
- HisTrap FF and GSTrap FF columns.
2.3 Phosphorylation
of Bacterially
Expressed AMPK at
Thr172 Using CaMKK2
- Gel filtration equilibration buffer: 50 mM Na HEPES, pH 8.0,
200 mM NaCl. - Dialysis buffer for phosphorylated, active AMPK: 50 mM Na
HEPES, pH 8.0, 200 mM NaCl, 50% (v/v) glycerol. - Protein purification system (e.g., GE Healthcare A ̈KTA).
- GSTrap FF and HiLoad 16/600 Superdex 200 pg columns.
- Dialysis tubing or cassettes.
- Unlabeled Mg.ATP: 10 mM MgCl 2 , 400μM unlabeled ATP.
2.4 In-Solution
Kinase Assay
- 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). - 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.