Sep 15, 2022
Fractionation of synaptosomes
- Chuyu Chen1,
- Ciarra Smith1,
- Loukia Parisiadou1
- 1Northwestern university
Protocol Citation: Chuyu Chen, Ciarra Smith, Loukia Parisiadou 2022. Fractionation of synaptosomes. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9d85zg3e/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: September 15, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70094
Keywords: ASAPCRN
Abstract
This protocol details a step by step method to prepare pure fractions of synaptosomes for biochemical analysis.
Pre-chill the homogeniser and buffers on ice. Weigh the tissue
Add 4x volume of ice-cold homogenizing buffer to glass homogenizers
Apply 12 strokes of even tension with the homogeniser rod
Transfer to 1.5ml tube (original tube)
Determine which tissue has the smallest volume and use that volume for all samples
Centrifuge at 1,000g for 10mins
Transfer the supernatant into fresh 1.5ml tube and spin at 12,500g for 15mins
Discard supernatant carefully
Resuspend pellet in 1ml of H-buffer, transfer to fresh glass homogenizer and mash for 6 strokes even tension
In polypropylene/ultra-clear tube add 5ml of 1.2M sucrose
Slowly and steadily add in 5ml of 0.8M sucrose to create clear/distinct gradient
Overlay sample on top of gradient
Weigh sucrose gradient tubes (add H-buffer if weight is off by more than 0.05g)
Centrifuge with slow accleration and zero brake in 4 degrees at 23,600g for 70 mins in a SW41 rotor
Using syringe, extract synaptosome layer between 0.8M and 1.2M sucrose and transfer into fresh 1.5ml tubes
Plate sample with appropriate dilutions into 24-well plate (dilute with H-buffer or PBS)
Spin in 4°C, max speed ~4,000 rpm for 20mins
Remove liquid and fix in 4% PFA for 20mins on rocker/shaker for 20mins
Wash x2~3 with PNBS
Either cover and place in fridge or move directly to immunofluorescence protocol