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How to separate serum lipoprotein
1. Configuration of the NaBr gradient: â— Analytically pure NaBr must be used with very dry. NaBr crystals were stored in an oven at 210 ° C overnight before use, and stored in a desiccator after removal. â— Configuration density: 1.006 g / ml, 9 g / l 1.016 g / ml, 27 g / l 1.063 g / ml, 95 g / l 1.210 g / ml , 283.4g / l 1.368g / ml, 524.8g / l â— Configure NaBr solvent 0.05% EDTA (pH 7.0), EDTA must be filtered before use, stored in a brown bottle, 4 ° C, the density should be measured before use ( Use Abbe refractometer or other constant temperature refractometer (concentration-density configuration table see below) 2. Separation step: I. Removal of blood cells: 1000xg, 20 points II. Removal of chylomicron (see Technical Lectures) III. Serum/1.386 G/m solution = 1:4 ratio mixed sample liquid for separation (Example: P40ST horizontal rotor with 0.2ml serum plus 0.8ml 1.386g/ml, NaBr solution; P35ZT zone with centrifuge rotor with 200ml serum plus 800ml 1.386g /ml NaBr solution) IV. Gradient laying: If using a 13 ml centrifuge tube, add 1 ml of the mixed sample solution at the bottom, and then lay down the layer: 3.2 ml, 1.21 g/ml NaBr; 3.8 ml 1.063 g/ml NaBr solution; Ml, 1.016g/ml NaBr solution; 1.2ml 1.006g/ml NaBr solution. If using P35ZT zone with rotor, first measure from low to high density from rotor Add 1.006, 1.016, 1.063, 1.21 gradient solution and finally add the sample mixture, the reference capacity is 74ml, 193ml, 228ml, 195ml and 1000ml. 3. Centrifugal process: â— P40ST rotor: 38,500rpm (263,000xg), 24hrs, 14°C, accelerate “8â€, decelerate “7†◠P35ZT zone with rotor: 35,000rpm (122,000xg), 49hrs, 14°C