Jun 15, 2023
Endosome isolation
- 1Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Chuyu Chen 2023. Endosome isolation . protocols.io https://dx.doi.org/10.17504/protocols.io.14egn3bp6l5d/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: June 15, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 83503
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: ASAP-020600
Abstract
Subcellular fractionation to isolate early and late endosomes (EEs and LEs) by performing a series of centrifugation steps.
Continuous sucrose gradient tube preparation
Continuous sucrose gradient tube preparation
prepare 0.5M and 2M sucrose buffer
prepare buffer S2 by mixing 0.5M and 2M sucrose at 1:2 and buffer S3 at 2:1 ratio
place Beckman ultracentrifuge tube (No.344059) on dry ice
add 2.5ml 2M sucrose in the tube and wait for 00:10:00 or until it frozen.
10m
add 2.5 ml buffer S2 on top of frozen 2M sucrose and wait for 00:10:00 or until it frozen
10m
add 2.5 ml buffer S3 on top of frozen buffer S2 and wait for 00:10:00 or until it frozen
10m
add 2.5 ml 0.45M sucrose on top of frozen buffer S3 and wait for 00:10:00 or until it frozen
10m
keep the frozen tubes at -80 until experiment day
place the frozen tubes at 4C for 05:00:00 or until defrosted before use
5h
Fractionation
Fractionation
Dissect striatum from mice, fresh freeze the tissue with dry ice
Prepare homogenization buffer (HB): 150mM Nacl, 10mM HEPES, 1mM EGTA, 0.1M MgCl
Homogenize striatum tissue with 1ml HB containing 1x Halt protease and phosphatase inhibitor (EDTA-free) by Dounce homogenization on ice (30 strokes)
Centrifugation 00:10:00 , 3,000g, 4C
10m
load postnuclear supernatants onto the top of continuous sucrose gradient
Centrifugation 18:00:00 , 200,000g, 4C (Accel and Decel -> slow)
18h
Collect fractions from top to bottom of the gradient
TCA precipitation
TCA precipitation
50m 3s
50m 3s
add equal volume of ice-cold 55% TCA and incubate on ice for 00:30:00
30m
centrifugation 00:10:00 , 20,000g, 4C
10m
wash pellet with 1ml ice-cold acetone centrifugation 00:10:00 , 20,000g, 4C
10m
go to step #19 for 2 times. Total 3 washes
resuspend pellet with 70ul of 1% SDS buffer (1% SDS, 10mMTris, 2mMEDTA) containing 1x Halt protease and phosphatase inhibitor (EDTA-free), sonication for 00:00:03 and followed by incubation on orbital shaker for 1hr at room temperature.
3s
Store samples at -80 for western blot assay