- Wash buffer: 50 mM HEPES-NaOH pH 7.4, 1 mM EGTA,
10 mM MgCl 2 , 0.1% (v/v) Tween 20, 0.01% (w/v) BSA. - Kinase assay reagents: 100 mM ATP, 3000 Ci/mmol (10 mCi/
ml)^33 P–ATP, 2% (v/v) phosphoric acid, and 20 mM SAMS
peptide. - AMPK activators: 50 mM PF-06409577, a proprietary Pfizer
AMPK activator. - Negatively-charged phosphocellulose (PH) filter plate.
2.5 Surface Plasmon
Resonance
- SPR buffer A: 25 mM Tris–HCl pH 7.5, 150 mM NaCl,
250 μM TCEP, 0.01% (v/v) P20 surfactant, 0.2 mg/ml BSA,
150 μM AMP (seeNotes 11and 12 ). - SPR buffer B: Buffer Aþ2% (v/v) DMSO.
- HBS-P buffer: 10 mM HEPES-NaOH, pH 7.5, 150 mM
NaCl, 0.01% (v/v) P20. - SPR instrument: e.g., Biacore™3000.
- Chips for immobilization of protein: Streptavidin-based sensor
chips. - Conditioning solution: 1 M NaClþ50 mM NaOH.
- Software for SPR data analysis: e.g., Scrubber2 and BIAeval.
3 Methods
Carry out all procedures at room temperature unless otherwise
specified.3.1 Recombinant
Protein Production
For bacterial expression of AMPK and purification, followsteps 1– 14
below.- For bacterial expression of AMPK, design a tricistronic AMPK
expression plasmid (seeNote 2) (Fig.1a). - Synthesize the above-described gene with codon optimization
for bacterial expression. - Subclone the tricistronic gene into theNcoIandXhoIsites of
bacterial expression vector (pET14b) under the control of T7
promoter using standard molecular biology techniques (see
Note 13). - Transform the construct intoE. colicells and select transfor-
mants on LB agar plates containing ampicillin (100μg/ml). - For large-scale expression and purification, inoculate an Erlen-
meyer flask containing 1 l of LB broth supplemented with
100 μg/ml ampicillin with 25 ml of overnight culture and
grow in a shaker incubator at 37CtoanOD 600 of 0.8.
Biophysical Studies to Evaluate Protein-Ligand Interactions 35