Purifiying CCVs and clathrin from Rat Brain.
(from Ian Mills, adapted from Pearse & Robinson, EMBO J. 9:1951-7 (1984))
1. Wash 10 rat brains in HKM buffer (25mM Hepes pH 7.4, 125mM potassium acetate, 5mM magnesium acetate + 1mM DTT).
2. Make up to 40ml with HKM.
3. Add Calbiochem protease cocktail set III.
4. Homogenise brains in a potter homogeniser
5. Spin at 7000rpm for 20mins in an SS34 rotor.
6. Collect supernatant.
7. Ultracentrifuge at 45000rpm for 40 minutes in a 70Ti rotor.
8. Resuspend pellet in about 10ml of HKM.
9. Homogenise pellet.
10. Add an equal volume of HKM containing Ficoll (12.5% w/v) and sucrose (12.5% w/v). Mix by inversion to ensure homogeneity.
11. Spin in a 70Ti rotor at 25000rpm for 20 minutes.
12. Dilute the supernatant 1:5 in HKM.
13. Centrifuge at 35000rpm for 60 minutes in a 45Ti rotor.
14. Resuspend pellet in 15ml of HKM and homogenise.
15. Leave on ice for about one hour.
16. Spin at 13000rpm for 10 minutes to sediment insoluble ...
Refer a Friend
Link To Us
Bookmark Us