Analysis of Lipid Droplets in Cardiac Muscle - Methods in Cell Biology - page 129

3. NaCl (Sigma-Aldrich, cat. no. S7653)
4. KCl (Sigma-Aldrich, cat. no. P9333)
5. Na 2 HP0 4 (Sigma-Aldrich, cat. no. S5136)
6. KH 2 PO 4 (Sigma-Aldrich, cat. no. P5655)
7. Sodium dodecyl sulfate (Sigma-Aldrich, cat. no. L3771)

8.1.1.2 Buffers
1. Tris-EDTA-sucrose buffer (10 mM Tris, pH 7.4, 1 mM EDTA) supplemented
the day of the experiment with 1 tablet of protease inhibitor cocktail for 50 ml
buffer and 10% final sucrose.
2. PBS, pH 7.4.
3. 2 concentrate Laemmli sample buffer containing 4% SDS, 20% glycerol, 10%

2-mercaptoethanol, 0.004% bromphenol blue, and 0.125 M Tris HCl, pH

6.8

(Sigma-Aldrich, cat. no. S3401).

8.1.2 Equipment
1. Refrigerated table top Microfuge (Beckman 22R)
2. Refrigerated Centrifuge (Beckman Allegra 22X-R)
3. Ultracentrifuge (Bekman Optima L 90K)
4. 200 m l pipet tips with large orifice (VWR, cat. no. 53503-612)
5. SW55 Ti rotor (Beckman Coulter, cat. no. 331302)
6. 5 ml Ultracentrifuge Tube (Beckman, cat. no. 326819)
7. 1 ml syringe (BD, cat. no. 309602) and 27G1/2 needle (BD, cat. no. 305109) cut
to provide a blunt end
8. Glass Dounce homogenizer and fitted pestle (Wheaton, cat. no. 357542)
9. 50 ml conical centrifuge tube (Corning, cat. no. 430828)
10. Liquid nitrogen cryogenic container
11. Biosqueeze cryogenic tissue clamping (Fisher Scientific, cat. no. 200489)
12. 4-12% pre-poured gels for protein analysis (Life Technologies, CA, cat. no.
NP0330)
13. Nitrocellulose membrane (GE Healthcare, NJ, cat. no. RPN203D)
14. Enhanced chemiluminescence (ECL) system (GE Healthcare, cat. no.
RNP2232)
15. Super Signal West Dura (Pierce Biotechnology, cat. no. 37071) or West Femto
(Pierce Biotechnology, cat. no. 34094)

8.1.3 Experimental design
1. Mice are euthanized by cervical dislocation, heart tissue is then excised and
immediately snap-frozen using cryogenic tissue clamps. Frozen tissues are
quickly weighed, reduced to powder using a small ceramic mortar cooled with
liquid nitrogen, and maintained on dry ice. Frozen powered tissues are carefully
collected and set in a glass bounce homogenizer (ratio 100 mg heart tissue/1 ml
ice-cold Tris-EDTA buffer pH 7.4 with cocktail inhibitors) on ice.