- Heating/cooling shaking incubator.
- IPTG 1000stock: 500 mM isopropylβ-D-1-thiogalactopyr-
anoside (IPTG) in water. - Spectrophotometer that can measure wavelengths of 600 nm.
- Plastic cuvettes suitable for spectrophotometer.
- Centrifuge.
- Protease inhibitor stock: 10 mM leupeptin, 1 M benzamidine-
HCl, and 100 mM AEBSF, all solubilized in water. - Cell disruptor or homogenizer or sonicator.
- Nickel column: Prepacked 5 ml nickel Sepharose IMAC
(immobilized metal affinity column) resin. - Peristaltic pump.
- Lysis buffer: 50 mM Tris–HCl, pH 7.6, 500 mM NaCl, 5%
glycerol, 50 mM imidazole, 2 mMβ-mercaptoethanol. - Elution buffer: 50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 10%
glycerol, 2 mMβ-mercaptoethanol, 400 mM imidazole. - SEC column: S200 size-exclusion chromatography column,
120 ml bed volume. - Storage buffer: 50 mM Tris–HCl, pH 8.0, 150 mM NaCl,
2 mM Tris (2-carboxyethyl) phosphine (TCEP). - 15 ml concentrator, 30 kDa cutoff.
- AMP: 100 mM stock (solubilized in 50 mM Tris–HCl,
pH 7.4) for CaMKK2 treatment. - ATP: 100 mM stock (solubilized in 50 mM Tris–HCl, pH 7.4)
for CaMKK2 treatment. - MgCl 2 : 1.0 M magnesium chloride (dissolved in water) for
CaMKK2 treatment. - CaMKK2 (seeSubheadings2.2 and 3.2).
2.2 Production
and Purification
of CaMKK2 for AMPK
Phosphorylation
- CaMKK2 expression vector: CaMKK2 cDNA (UniProt:
Q96RR4-1) in pFASTBAC1-FLAG-N-TEV. - Sf21 insect cells.
- Sf-900 II media.
- Heating/cooling shaking incubator.
- PBS (phosphate-buffered saline): 137 mM NaCl, 2.7 mM KCl,
10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4. - Insect lysis buffer: 50 mM Tris–HCl, pH 7.4, 200 mM NaCl,
and 1 mM EDTA. - FLAG monoclonal antibody-coupled affinity resin.
- Insect elution buffer: Insect lysis buffer with FLAG peptide
(DYKDDDDK) added (0.25 mg/ml). - Liquid nitrogen.
Visualization of Drug Binding Sites 19