Sedimentation Assay
Michael X. Henderon
Abstract
This protocol details sedimentation assay for alpha-synuclein fibrils.
Attachments
Steps
With Sucrose Cushion
Dilute 4µL of 5mg/mL α-synuclein PFFs to 40µL in PBS in ultracentrifuge tubes.
Add 40µL 20% sucrose beneath PFFs.
Ultracentrifuge at 100000x g,0h 0m 0s (45000rpm,0h 0m 0s) for 0h 30m 0s at 22°C.
Remove 70µL supernatant and add to new tube.
Add 60µL 12.5% sucrose to the pellet and resuspend the pellet by pipetting.
Dilute samples in 5x sample buffer.
Boil samples at 95°C for 0h 5m 0s.
Run samples on a 15% polyacrylamide gel.
Stain with Coomassie blue.
Without Sucrose Cushion
Dilute 4µL of 5mg/mL α-synuclein PFFs to 40µL in PBS.
Ultracentrifuge at 100000x g,0h 0m 0s (45000rpm,0h 0m 0s) for 0h 30m 0s at 25°C.
Remove supernatant and dilute in 5x sample buffer.
Add 40µL PBS to the pellet.
Resuspend pellet by pipetting up and down. Dilute in 5x sample buffer.
Boil samples at 95°C for 0h 5m 0s.
Run samples on a 15% polyacrylamide gel.
Stain with Coomassie blue.
