By JOHN M. GRAHAM
A very good e-book with lots of protocols for these operating with membranes. very good for subcellular fractionation of tissues.
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Extra info for Biomembrane Protocols: I. Isolation and Analysis
8. Since the pellet tends to slide as the meniscuspassesover it, keep the tip of the pipet or cannula close to the wall of the tube away from the pellet. 9. Method adapted from Fleischer et al. (12). 10. A fixed-angle rotor is used for the barrier separation to facilitate the resuspension of the upper pellet without disturbing the nuclei. 11. If brand-new polycarbonate tubes are used for the barrier separation, the lower part of the pellet (nuclei) may tend to slide off the wall of the tube during resuspension.
07 mm) attached to an overhead electric (thyristor-controlled) motor. 2. A loose-fitting Dounce homogenizer (30 mL). 3. 20-mL Syringe fitted with a wide-bore metal cannula (l-2 mm id). 4. 50-mL Conical-bottomed polypropylene tubes for a refrigerated lowspeed centrifuge (swing-out rotor); 50-mL polycarbonate tubes for a refrigerated high-speed centrifuge (fixed-angle rotor). 5. 4. 6. 2. “Heavy” Mitochondrial Fraction by Density Barrier 1. , item 1). 2. , item 2). 3. , item 4). 4. 5. 5. 5, 1 mM MgC12.
5. 20-mL Syringe. 3. Purification of Nuclear Pellet in a Nycodenz Gradient 1. , item l), and prepare solutions of 20, 30, 35,40, and 50% by dilution with TKM. 2. Loose-fitting Dounce homogenizer (about 5-mL capacity). 3. Polycarbonate tubes of about 14-mL capacity to fit a high-speed or ultracentrifuge swing-out rotor. 4. Refractometer. 4. Purification of Nuclear Membrane in High-Ionic-Strength Medium 1. 5, 1 miV PMSF, 1 mM EGTA. 2. 5, 1 mMPMSF, 1 miVEGTA. 2M NaCl m Buffer 2 4. P-Mercaptoethanol.
Biomembrane Protocols: I. Isolation and Analysis by JOHN M. GRAHAM