AMPK Methods and Protocols

2.3 Crystallization
for ADaM Site
Activators

1. AMP: 9 mM adenosine monophosphate (AMP) solubilized in
   50 mM Tris–HCl, pH 7.4.


2. A769662: 3 mM A769662 solubilized in DMSO.


3. Staurosporine: 3 mM staurosporine solubilized in DMSO.


4. S108tide: 600μM S108 synthetic peptide (corresponding to
   β1(102–114) Arg112–114) (NH 2 -KLPLTRSHNNFVARRR-
   COOH), solubilized in 50 mM Tris–HCl, pH 7.4.


5. SAMS peptide: 600 μM SAMS peptide (NH 2 -
   HMRSAMSGLHLVKRR-COOH), solubilized in 50 mM
   Tris–HCl, pH 7.4.


6. 24-Well crystallization plate.


7. Reservoir solution: 7–8.5% polyethylene glycol (PEG) 3350,
   0.1 M MgCl 2 , 1.0% glucose, 0.0005–0.003% cocamidopropyl
   betaine, and 0.1 M imidazole HCl (pH 6.2).

2.4 Crystallization
for C2-Site Activators

1. C2: 9 mM C2 (5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phos-
   phonic acid) solubilized in DMSO.

2.5 Cryoprotection
of AMPK Crystals

1. 1% cryo-solution: 7–8.5% polyethylene glycol (PEG) 3350,
   0.1 M MgCl 2 , 1.0% glucose, 0.0005–0.003% cocamidopropyl
   betaine and 0.1 M imidazole (pH 6.2), 1.0% glycerol, 1.0%
   PEG 400, 1.0% ()-2-methyl-2,4-pentanediol (MPD), 1.0%
   sucrose, 1.0% sorbitol, and 1.0% ethylene glycol.


2. 2% cryo-solution: 7–8.5% polyethylene glycol (PEG) 3350,
   0.1 M MgCl 2 , 1.0% glucose, 0.0005–0.003% cocamidopropyl
   betaine and 0.1 M imidazole (pH 6.2), 2.0% glycerol, 2.0%
   PEG 400, 2.0% ()-2-methyl-2,4-pentanediol (MPD), 2.0%
   sucrose, 2.0% sorbitol, and 2.0% ethylene glycol.


3. 3% cryo-solution: 7–8.5% polyethylene glycol (PEG) 3350,
   0.1 M MgCl 2 , 1.0% glucose, 0.0005–0.003% cocamidopropyl
   betaine and 0.1 M imidazole (pH 6.2), 3.0% glycerol, 3.0%
   PEG 400, 3.0% ()-2-methyl-2,4-pentanediol (MPD), 3.0%
   sucrose, 3.0% sorbitol, and 3.0% ethylene glycol.


4. 4% cryo-solution: 7–8.5% polyethylene glycol (PEG) 3350,
   0.1 M MgCl 2 , 1.0% glucose, 0.0005–0.003% cocamidopropyl
   betaine and 0.1 M imidazole (pH 6.2), 4.0% glycerol, 4.0%
   PEG 400, 4.0% ()-2-methyl-2,4-pentanediol (MPD), 4.0%
   sucrose, 4.0% sorbitol, and 4.0% ethylene glycol.


5. 5% cryo-solution: 7–8.5% polyethylene glycol (PEG) 3350,
   0.1 M MgCl 2 , 1.0% glucose, 0.0005–0.003% cocamidopropyl
   betaine and 0.1 M imidazole (pH 6.2), 5.0% glycerol, 5.0%
   PEG 400, 5.0% ()-2-methyl-2,4-pentanediol (MPD), 5.0%
   sucrose, 5.0% sorbitol, and 5.0% ethylene glycol.


6. Liquid nitrogen.

20 Christopher G. Langendorf et al.