SV 2 subpopulation PROTOCOL.
9-9-03
1. 2 rat brains were homogenized in buffer A (0.32 M sucrose, 10 mM HEPES, pH 7.2)
2. Spin for 3 min, 1000g
3. Spin supernatant for 15 min, 11000g
4. Resuspend pellet in 4 ml final volume and load on 8 ml of 7.5% Ficoll
5. Spin in Beckman SW40 rotor at 22.5K, 40 min
6. Remove supernatant by aspiration and resuspend the pellet by gentle pipetting with 5
ml Gilson using 4 ml PBS. Gentle pipetting is for preservation of synaptosome integrity
at this stage of the protocol. Transfer the resuspended material to 15 ml Falcon, top it up
to 14 ml with PBS and spin 5 min, 1500g
7. Disrupt pelleted synaptosomes by adding 5 ml 10 mM HEPES, pH 7.2, 0.1 mM
Na2EGTA and rough pipetting (don't care about foaming).
8. Top it to 8 ml and add 1 ml 1.4 M Kgluconate to bring ionic strength to around
physiological values.
9. Spin 20 min, 10000 rpm in SW40
10. Withdraw 7.6 ml of supernatant without touching the pellet into 15 ml falcon and add
4.4 ml of Optiprep. Mix and transfer to the NVT centrifugation tubes.
11. Spin 1.5 hrs, 65K in a near vertical Beckman rotor NVT65. Note, I used only the
near-vertical rotor and I don't know whether the gradient separation of vesicle
subpopulations can be achieved in other types of rotors.
12. Fractionate the formed gradient by inserting a glass capillary from top to the bottom
of the centrifugation tube and pumping the gradient slowly out into a fraction collector
(1ml fractions).
Notes: All solutions should be cold. Use OptiSeal polyallomer centrifuge tubes for
NVT65: 16x67mm with plugs, order No 362181