- Nontreated 24-well plates.
- Bicinchoninic acid protein assay reagent from Pierce.
- Synthetic SAMS peptide (HMRSAMSGLHLVKRR).
- [γ-^32 P]ATP 3000 Ci/mmol, 10 mCi/ml.
- Assay buffer (5): 20 mM HEPES (pH 7), 400 mM NaCl,
5 mM DTT, 25 mM AcMg2+, 1 mM SAMS peptide,
1 mM AMP. - Hot ATP (5), for 200 ml: 10 ml of ATP 3000 Ci/mmol,
10 ml mCi/ml; 40 ml of 10 mM ATP (cold, in 25 mM Tris,
pH 8.0), H 2 O up to 200 ml. - Whatman P81 paper (2 cm^2 square pieces, labeled)
- Orthophosphoric acid.
- Acetone.
- Scintillation liquid or water.
- Scintillation vials.
- Scintillation counter.
Western Blot of AMPK Targets
- Isolated islets.
- Benchtop microcentrifuge (1.5 ml tubes).
- Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl,
10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4. - RIPA buffer: 150 mM NaCl, 10 mM Tris, pH 7.2, 5 mM
EDTA, 0.1% (w/v) SDS, 1% (w/v) sodium deoxycholate, 1%
(v/v) Triton X-100, protease and phosphatase inhibitors
cocktails. - 6SDS sample buffer: 375 mM Tris–HCl, pH 6.8, 12% (w/v)
SDS, 60% (v/v) glycerol, 0.6 M DTT, 0.06% (w/v)
bromophenol blue. - Mini-Protean electrophoresis system (BioRad).
- 8% polyacrylamide gels.
- PVDF membrane.
- Transfer buffer: 25 mM Tris-base, 192 mM glycine, and 10%
(v/v) methanol. - Blocking solution: PBS, 0.1% (v/v) Tween 20, 5% (w/v) semi-
skimmed milk. - Washing buffer: PBS, 0.1% (v/v) Tween 20.
- Antibodies: anti-ACC (3662, cell signaling), phospho-ACC
(Ser79) (3661, cell signaling), Raptor (2280, cell signaling),
phospho-raptor (Ser792) (2083, cell signaling), AMPKα
(2532, cell signaling), phospho-AMPKα(Thr172) (2535, cell
signaling), goat anti-rabbit IgG-HRP (AB6721, Abcam).
Manipulation and Measurement in Islets 417