Nov 12, 2023
Preparation of an Enriched Synaptic Vesicle Fraction
- 1University of California, San Diego
Protocol Citation: Leonardo A Parra-Rivas 2023. Preparation of an Enriched Synaptic Vesicle Fraction. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4qr62vo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 12, 2023
Last Modified: November 12, 2023
Protocol Integer ID: 90817
Abstract
Preparation of an Enriched Synaptic Vesicle Fraction
Mouse brain tissue was homogenized in a buffer containing 0.32 M sucrose in PBS, supplemented with protease/phosphatase inhibitors.
The homogenate was centrifuged at 1,000 × g for 10 min at 4 °C to
remove nuclei and cellular debris (P1). The supernatant (S1) was centrifuged at
15,000 × g for 15min at 4 °C to yield a crude synaptosomal pellet (P2).
This pellet was hypo-osmotically lysed in water containing protease inhibitors for 5 min at 4
°C and passed through both a 22- and 27½-gauge needle (10 times each).
This suspension was centrifuged at 23,000 × g for 22 min at 4 °C, and the resulting supernatant LS1(S3) was centrifuged again at 174,900 × g for 2 hours at 4 °C in a STi32 Beckman rotor. The final pellet (LP2 or P4) containing an enriched fraction of synaptic vesicles was used for subsequent
experiments.