Ja 20 fixed angle rotor

The tissue or packed cell membranes are solubilized in 25 volumes buffer containing 1.25% (w/v) cholate, 500 mM NaCl and 100 mM sodium phosphate buffer pH 7.4 (to give 1.2% (w/v) cholate in the final mixture) and 1 mM phenylmethanesulfonyl fluoride (PMSF; CAS 329-98-6) as protease inhibitor (optional). After incubation (10 min, 0 °C), the mixture is centrifuged (Beckmann JA-20 fixed angle rotor, 39,000×g, 18,000 rpm, 20 min, 4 °C). The supernatant is mixed with 1.5 volumes of the above reconstitution mixture, incubated (15 min, 0 °C) and gel filtrated on spin columns (see below) to remove detergent and replace the reconstitution buffer with the desired internal medium. This means that the reconstitution mixture contained about 15 times more lipids than the endogenous lipids present in the rat of mouse brain extracts.

Fresh liver was taken from mice, and homogenized in 250 mM sucrose, 10 mM HEPES, 1 mM EGTA. 0.5% fatty acid free bovine serum albumin (BSA) pH 7.4 at 4°C, with seven passes of a loose glass Dounce homogenizer (Type A). Homogenates were centrifuged in glass tubes at 2900 rpm (1000 g) for 10 min in a pre-chilled 4°C Beckman centrifuge (JA-20 Fixed angle rotor). The supernatant was then centrifuged in glass tubes at 8500 rpm (8700 g) for 10 min at 4°C. The supernatant was aspirated and any visible lipid was carefully removed from the sides of the tubes. The pellet was washed with 5 mL of mIR-05 buffer (0.5 mM EGTA, 3 mM MgCl₂, 20 mM taurine, 10 mM KH₂PO₄, 20 mM HEPES, 110 mM sucrose, 1 mg/ml fatty acid free BSA, pH 7.2), and centrifuged at 8500 rpm (8700 g) for 10 min at 4°C. After aspiration and removal of visible lipid, the pellet was suspended in 1ml of mIR-05 buffer and kept on ice until used. All measurements were taken within two hours of preparation. Protein concentration was determined using the DC-Protein Assay (BioRad) as per manufacturers instruction.