Reagents and equipment:
1. Yeast protoplasts are prepared in sorbitol buffer;
2. NycoprepTM Universal (600g/L Nycodenz);
3. Nycodenz â—‹ R working solution: 500g / L Nycodenz;
4. Sorbitol buffer: 0.6 mol / L sorbitol, 20 mmol / L Mes-KOH, pH 6.0;
5. Sorbitol buffer (2×): 1.2 mol/L sorbitol, 40 mmol/L Mes-KOH, pH 6.0;
6. Suspension buffer: 0.6 mol / L sorbitol, 20 mmol / L Mes-KOH, pH 7.4;
7. Add protease inhibitors to the solution as required;
8. High-speed centrifuge with 8×50ml fixed angle rotor;
9. Ultracentrifuge with a 12-13ml tube bucket rotor;
10. Dounce homogenizer (about 40ml tightly prepared, Wheaton A type);
11. Dounce homogenizer (about 40ml loosely formulated, Wheaton B type);
experimental method:
All operations must be carried out at 0-4 °C.
1. Homogenize the protoplasts by grinding 15 times with a mortar using a compact Dounce homogenizer;
2. Dilute with an equal volume of sorbitol buffer and centrifuge at 1500g for 5min with a fixed angle rotor;
3. Gently pour off the supernatant and retain the precipitate;
4. Resuspend the pellet to the original volume with sorbitol buffer and repeat steps 1 and 2.
5. Combine all supernatants and centrifuge at 12000g for 10min;
6. Resuspend the crude mitochondrial pellet in approximately 40 ml of sorbitol buffer and gently grind several times with a loosely prepared Dounce homogenizer;
7. Centrifuge at 1500g for 5min to remove remaining core and debris;
8. Remove the supernatant with a syringe with a metal trocar, be careful not to stir the loose deposits;
9. 12000 g of the supernatant was centrifuged for 10 min, and the pellet was resuspended in about 4 ml of sorbitol buffer (using a loosely prepared Dounce homogenizer);
10. Prepare two 180g/L and 145g/L Nycodenz solutions from 25ml sorbitol buffer (2×) and 18ml (and 14.5ml) 500g/L Nycodenz working solution; prepare each solution to 50ml with water;
11. Load 5 ml of each of these Nycodenz solutions and approximately 2 ml of a crude mitochondrial suspension (filled with sorbitol buffer if necessary) in a tube suitable for a bucket rotor;
12. Centrifuge at approximately 200000 g for 30 min. Purified mitochondria are banded at the interface of two Nycodenz solutions;
13. Collect the zone with a syringe with a metal trocar; dilute with 4 volumes of suspension buffer; centrifuge at 12000g for 15 min, resuspend the pellet in suspension buffer;
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