How to separate the cell membranes of male rats using a centrifuge?

Release time:

2015-05-13

Overview:

Wash the centrifuged precipitate with a 0.25 mol/L sucrose solution, centrifuge at 1000 g (3500 rpm) for 10 min, and repeat once.

Reagents: physiological saline, 0.25 mol/L sucrose solution (containing 0.3 mol/L CaCl₂), 0.34 mol/L sucrose solution (containing 0.3 mol/L CaCl₂); Equipment: a standard centrifuge for isolating cell membranes.

1) Take male mice (150 g) that have been fasted for 12 hours. Decapitate them to euthanize, then bleed them. Dissect out the liver, quickly rinse it with physiological saline, and blot dry with filter paper. Take 3 g of liver tissue, mince it thoroughly, and wash it several times with cold 0.25 mol/L sucrose solution. Homogenize it in 18 ml of pre-cooled 0.25 mol/L sucrose solution, then filter through a double-layer nylon membrane and set aside for later use.

2) Transfer the homogenate into a centrifuge tube, carefully add an equal volume of 0.34 mol/L sucrose solution along the tube wall to cover the sample, and centrifuge at 700g for 10 minutes.

3) Wash the centrifuged precipitate with a 0.25 mol/L sucrose solution, centrifuge at 1000 g (3500 rpm) for 10 min, and repeat once.

4) After centrifugation, the precipitate consists of cell nuclei, with a relatively loose upper layer above it. Carefully aspirate this upper layer and suspend it in a sucrose solution with a density of d20 = 1.16 (51 g/L). Transfer the suspension into a centrifuge tube and carefully overlay it along the tube wall with a sucrose solution of density d20 = 1.18 (51.5 g/L), which will form a layer on top of the sample. Centrifuge at 700 g (3000 rpm) for 10 minutes. The cellular membrane fraction will accumulate at the interface between the two layers of solution. Carefully aspirate this fraction and store it at low temperature for further analysis.

What is a centrifuge tube adapter?

A method for separating plant cell membranes using a high-speed centrifuge