Jan 24, 2024

Sedimentation Assay

DOI
https://dx.doi.org/10.17504/protocols.io.eq2lyjb9mlx9/v1
  • Michael X. Henderon1
  • 1Van Andel Institute
Michael Henderson
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DOI: https://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: September 20, 2024
Protocol  Integer ID: 93623
Keywords: ASAPCRN, synuclein fibril, protocol details sedimentation, sedimentation, synuclein, fibril
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-020616
Abstract
This protocol details sedimentation assay for alpha-synuclein fibrils.
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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 4 µL of 5 mg/mL α-synuclein PFFs to 40 µL in PBS in ultracentrifuge tubes.

Add 40 µL 20% sucrose beneath PFFs.

Ultracentrifuge at 100000 x g (45000 rpm ) for 00:30:00 at 22 °C .

30m
Remove 70 µL supernatant and add to new tube.

Add 60 µL 12.5% sucrose to the pellet and resuspend the pellet by pipetting.

Dilute samples in 5x sample buffer.
Boil samples at 95 °C for 00:05:00 .

5m
Run samples on a 15% polyacrylamide gel.

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


Stain with Coomassie blue.
Without Sucrose Cushion
35m
Dilute 4 µL of 5 mg/mL α-synuclein PFFs to 40 µL in PBS.

Ultracentrifuge at 100000 x g   (45000 rpm ) for 00:30:00 at 25 °C .

30m
Remove supernatant and dilute in 5x sample buffer.
Add 40 µL PBS to the pellet.

Resuspend pellet by pipetting up and down. Dilute in 5x sample buffer.
Boil samples at 95 °C for 00:05:00 .

5m
Run samples on a 15% polyacrylamide gel.

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


Stain with Coomassie blue.