- Variable-temperature shaking incubator with fittings for
12 2 L conical flasks. - Centrifuges and rotors, e.g., Beckman-Coulter J6 with
JA-25.50 rotor and Beckman-Coulter J6-M1 with JS 4.2 rotor. - Sonicator fitted with microtip.
- Syringe filters 0.45μm.
- CompetentE. coliBL21 Star (DE3).
- Stock solution of ampicillin (100 mg/mL).
- 14 Luria-Bertani broth (LB) agar plates (with ampicillin
100 μg/mL). - 250μL SOC media.
- 10 L of Luria-Bertani broth (LB).
- IPTG: 100 mM isopropylβ-D-galactosidase (IPTG) in water.
- Lysis buffer: 50 mM Tris–HCl pH 8.0, 300 mM NaCl, 1 mM
Tris(2-carboxyethyl)phosphine (TCEP), 20 mM imidazole,
1 mM MgCl 2 , 4 EDTA-free protease inhibitor tablets
(Roche Products Ltd), and 12μL benzonase.
200 mL of lysis buffer is needed for washing pellets from
12 0.8 L growth flasks (seeNote 1). Each 0.8 L culture
typically yields 4–5 g of bacterial pellet. - Nickel A buffer: 50 mM Tris–HCl pH 8.0, 300 mM NaCl,
0.5 mM TCEP, and 20 mM imidazole. - Nickel B buffer: 50 mM Tris–HCl pH 8.0, 300 mM NaCl,
0.5 mM TCEP, and 400 mM imidazole. - Gel filtration buffer: 50 mM Tris–HCl pH 8.0, 300 mM NaCl,
and 0.5 mM TCEP.
2.2 Crystallization
Reagents
and Equipment
- 24- and 48-well crystallization plates (Hampton Research).
- Crystallization solution: 13% (w/v) polyethylene glycol (PEG)
3350, 0.1 M MgCl 2 , 1% (w/v) glucose, 0.15% (w/v) cocami-
dopropyl betaine (CAPB) in 100 mM imidazole at pH 6.2. - Crystallization solution: 12% (w/v) PEG 3350, 300 mM
guanidine-HCl in 100 mM piperazine-N,N^0 -bis(2-ethanesul-
fonic acid) (PIPES) buffer at pH 7.2. - 10 mM staurosporine in DMSO and 10 mM AMP in water.
- Ethylene glycol.
- Low-power (magnification 40) stereo microscope.
- Range of sizes (0.1–0.4 mm) 18 mm mounted cryoloop
(Hampton Research Ltd). - Liquid nitrogen for crystal freezing and storage.
AMPK Crystallisation 3