蔗糖密度梯度离心手册-Sucrose Gradient separation manual.pdf

Sucrose Gradient Separation Manual _______________________ A protocol optimized for the separation of mitochondrial OXPHOS proteins by discontinuous sucrose gradient separation. March 2005 Also available at /PDF/sg.pdf support@ Ver.#JM5.1 Contents Page I. Required reagents and equipment…………………………………… 2 II. Sample preparation…………………………………………………………… 3 III. Sample solubilization………………………………………………………… 4 IV. Sucrose gradient preparation…………………………………………… 5 V. Centrifugation of gradients and elution of fractions………… 7 VI. Sample analysis………………………………………………………………… 8 VII. Buffer recipes……………………………………………………………………. 9 VIII. Optimization steps and general tips…………………………………. 10 IX. Troubleshooting Guide……………………………………………………… 12 XI. Flowchart…………………………………………………………………………… 13 X. Notes…………………………………………………………………………………. 14 I. Required Reagents ü n-dodecyl--D-maltopyranoside (Lauryl maltoside, Anatrace) ü Phosphate buffered saline, PBS (section VII) ü Sucrose solutions 15, 20, 25, 27.5, 30 and 35 % ü Double distilled water ü Protease inhibitor cocktail (section VII) ü 13 x 51 mm polyallomer centrifuge tubes (Beckman 326819) Required Equipment ü Swing-out compatible ultracentrifuge and rotor (eg. Beckman SW50.1 ü pH meter, weighing balance and other standard lab equipment ü Laboratory benchtop microfuge ü Protein electrophoresis and Western blotting equipment.