Isolation of 3 endosomal fractions from rat livers

1. homogenization of livers (~ 70 g) with 3 15 s strokes with warring blendor followed by 6 strokes with glass-teflon homogenizer (half-speed) in 0.25 M sucrose (+ 100 µl of Pepstatin, DTT, Cocktail to 100 ml of sol.); 1:3 w/vol; H

2. centrifuge at 500g for 10 min (1550 rpm Jouan); discard pellet P1

3. centrifuge S1 (max. vol. 190 ml) at 3,500g for 15 min (5,200 rpm TST 28.38); discard pellet P2

4. centrifuge S2 at 12,000g for 20 min (11,400 rpm TFT 55.38); discard pellet P3

5. dilute 16 ml of S3 with 7.5 ml of isotonic Percoll (Percoll/2.5 M sucrose, 9:1, vol/vol; pH 7.4) per tube

6. centrifuge at 29,900g for 45 min (17,900 rpm TFT 55.38)

7. harvest gradient down to marker beads of density 1.062 mg/ml (red) S4 (approx. vol. = 12 ml/tube)

8. add 2 volumes of ice-cold 0.15 M NaCl

9. layer fractions onto 2 ml of 2.5 M sucrose

10. centrifuge at 17,800g for 45 min (11,500 rpm TST 28.38)

11. remove white endosome bands from sucrose cushions in a vol. of 1 ml or less per tube and raise density to ≈1.15 g/ml with 0.38 ml of 2.5 M sucrose/ml; S5

12. layer 2-4 ml of the endosome fractions at the bottom of discontinous sucrose gradients (made by layering successively 2 ml of each sucrose sol. with densities of 1.033, 1.074, 1.11, 1.13 g/ml)

13. centrifuge at 197,500g for 90 min ( 39,200 rpm TST 41.14) or at 104,000g for 170 min (27,800 rpm TST 28.38)

14. harvest each distinct pure white fluffy band at the interfaces*; measure sucrose conc. with refractometer and made isotonic by addition of water H1 M1 L1

15. centrifuge at 28,800g for 30 min (17,600 rpm TFT 55.38); discard supernatant; resuspend pellets in a small volume H2 M2 L2

*The endosome fraction of lowest density corresponds to Multi Vesicular Bodies, of intermediate density smaller lipoprotein-containig vesicles (0.3-0.4 µm), of highest density bow- or arc-shaped double bilayer membranous structures often with terminal swellings (perhaps a receptor-recycling compartment).

Belcher, J.D.; Hamilton, R.L.; Brady, S.E.; Hornick, C.A.; Jaeckle, S.;Schneider, W.J.; Havel, R.J. (1987) Isolation and characterization of three endosomal fractions from the liver of estradiol-treated rats; Proc.Nat.Acad.Sci. U.S.A.; Vol. 84, pp. 6785-6789

Hornick,C.A.; Hamilton, R.L.; Spaziani, E.; Enders, G.H.; Havel, R.J. (1985) Isolation and Characterization of Multivesicular Bodies from Rat Hepatocytes: An Organelle Distinct from Secretory Vesicles of the Golgi Apparatus; J. Cell Biol.; Vol. 100, pp. 1558-1569

solutions to be made up:

0.25 M sucrose	42.8 g / 500 ml
2.5 M sucrose	59.92 g / 70 ml
8% (w/vol) sucrose	3.3 g / 40 ml
18% (w/vol) sucrose	7.72 g / 40 ml
26% (w/vol) sucrose	11.52 g / 40 ml
31% (w/vol) sucrose	14.04 g / 40 ml
0.15 M NaCl	4.38 g / 500 ml
Percoll/2.5 M sucrose	9:1 (vol/vol); pH 7.4