AMPK Methods and Protocols

9. Heating/cooling shaking incubator.


10. IPTG 1000
    stock: 500 mM isopropylβ-D-1-thiogalactopyr-
    anoside (IPTG) in water.


11. Spectrophotometer that can measure wavelengths of 600 nm.


12. Plastic cuvettes suitable for spectrophotometer.


13. Centrifuge.


14. Protease inhibitor stock: 10 mM leupeptin, 1 M benzamidine-
    HCl, and 100 mM AEBSF, all solubilized in water.


15. Cell disruptor or homogenizer or sonicator.


16. Nickel column: Prepacked 5 ml nickel Sepharose IMAC
    (immobilized metal affinity column) resin.


17. Peristaltic pump.


18. Lysis buffer: 50 mM Tris–HCl, pH 7.6, 500 mM NaCl, 5%
    glycerol, 50 mM imidazole, 2 mMβ-mercaptoethanol.


19. Elution buffer: 50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 10%
    glycerol, 2 mMβ-mercaptoethanol, 400 mM imidazole.


20. SEC column: S200 size-exclusion chromatography column,
    120 ml bed volume.


21. Storage buffer: 50 mM Tris–HCl, pH 8.0, 150 mM NaCl,
    2 mM Tris (2-carboxyethyl) phosphine (TCEP).


22. 15 ml concentrator, 30 kDa cutoff.


23. AMP: 100 mM stock (solubilized in 50 mM Tris–HCl,
    pH 7.4) for CaMKK2 treatment.


24. ATP: 100 mM stock (solubilized in 50 mM Tris–HCl, pH 7.4)
    for CaMKK2 treatment.


25. MgCl 2 : 1.0 M magnesium chloride (dissolved in water) for
    CaMKK2 treatment.


26. CaMKK2 (seeSubheadings2.2 and 3.2).

2.2 Production
and Purification
of CaMKK2 for AMPK
Phosphorylation

1. CaMKK2 expression vector: CaMKK2 cDNA (UniProt:
   Q96RR4-1) in pFASTBAC1-FLAG-N-TEV.


2. Sf21 insect cells.


3. Sf-900 II media.


4. Heating/cooling shaking incubator.


5. PBS (phosphate-buffered saline): 137 mM NaCl, 2.7 mM KCl,
   10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4.


6. Insect lysis buffer: 50 mM Tris–HCl, pH 7.4, 200 mM NaCl,
   and 1 mM EDTA.


7. FLAG monoclonal antibody-coupled affinity resin.


8. Insect elution buffer: Insect lysis buffer with FLAG peptide
   (DYKDDDDK) added (0.25 mg/ml).


9. Liquid nitrogen.

Visualization of Drug Binding Sites 19