Jan 24, 2024

Public workspaceSedimentation Assay

DOI
dx.doi.org/10.17504/protocols.io.eq2lyjb9mlx9/v1
  • Michael X. Henderon1
  • 1Van Andel Institute
Michael Henderson
Open access
DOI: dx.doi.org/10.17504/protocols.io.eq2lyjb9mlx9/v1
Protocol Citation: Michael X. Henderon 2024. Sedimentation Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyjb9mlx9/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: December 20, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 93623
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-020616
Abstract
This protocol details sedimentation assay for alpha-synuclein fibrils.
Attachments
932-2407.pdf
932-2407.pdf
360KB
With Sucrose Cushion
With Sucrose Cushion

Note
A sucrose cushion will minimize chances of the pellet being disturbed but may increase the chances of a-synuclein monomer ending up in the pellet fraction.

Dilute Amount4 µL of Amount5 mg/mL α-synuclein PFFs to Amount40 µL in PBS in ultracentrifuge tubes.

Pipetting
Add Amount40 µL 20% sucrose beneath PFFs.

Pipetting
Ultracentrifuge at Centrifigation100000 x g (Centrifigation45000 rpm ) for Duration00:30:00 at Temperature22 °C .

30m
Centrifigation
Remove Amount70 µL supernatant and add to new tube.

Pipetting
Add Amount60 µL 12.5% sucrose to the pellet and resuspend the pellet by pipetting.

Pipetting
Dilute samples in 5x sample buffer.
Boil samples at Temperature95 °C for Duration00:05:00 .

5m
Run samples on a 15% polyacrylamide gel.

Note
Amount20 µL or less of sample can be loaded. Loading less may make for a cleaner gel (no α-synuclein PFFs visible in the supernatant).


Pipetting
Stain with Coomassie blue.
Without Sucrose Cushion
Without Sucrose Cushion
35m
Dilute Amount4 µL of Amount5 mg/mL α-synuclein PFFs to Amount40 µL in PBS.

Ultracentrifuge at Centrifigation100000 x g   (Centrifigation45000 rpm ) for Duration00:30:00 at Temperature25 °C .

30m
Centrifigation
Remove supernatant and dilute in 5x sample buffer.
Add Amount40 µL PBS to the pellet.

Pipetting
Resuspend pellet by pipetting up and down. Dilute in 5x sample buffer.
Pipetting
Boil samples at Temperature95 °C for Duration00:05:00 .

5m
Run samples on a 15% polyacrylamide gel.

Note
Amount10 µL or less of sample can be loaded. Loading less may make for a cleaner gel (no α-synuclein PFFs visible in the supernatant).


Pipetting
Stain with Coomassie blue.