TITLE: α-Synuclein sedimentation assay 
AUTHORS: arpine.sokratian

[DESCRIPTION]
The protocol describes step-by-step instructions on how to set a sedimentation assay to monitor the aggregation of alpha-synuclein over time. The protocol allows for precise quantification of fibril formation kinetics using SDS-PAGE. This methodology enhances our understanding of the underlying mechanisms involved in protein misfolding and offers a reliable tool for investigating potential therapeutic interventions targeting alpha-synuclein aggregation.

[STEPS]
SECTION: Amplification set-up
1. Thaw down a-syn monomer and/or sonicated fibril aliquots. Use water-bath.
Do not generate bubbles by pipetting or shaking

SECTION: Amplification set-up
2. Measure monomer concentration via Nanodrop
Add Amount of  5x, 10x or 20x diluted aliquots in PBS onto nanodrop piedestal; 
Parameters: other proteins; coefficient extinction: 
5.98; MW: 14.4 kDA for human a-synuclein 
7.45; MW: 14.4 kDA for mouse a-synuclein
Perform two measurements for each dilution and confirm <10% standard error between two measurements
For fibril preparations, dilute in 6M guanidine and follow the aforementioned instructions derscribed for monomeric protein
SECTION: Amplification set-up
3. Calculate the volume of monomer need following:  0.3 mg/mL of monomer in a reaction (30 ul reaction/well). 
Dilute the monomer preparation in PBS to 2,5 mg/mL in order to filter the protein preparation to avoid any high mol. weight contaminants 
SECTION: Amplification set-up
4. Use  
to filter the diluted monomer concentration. Prior to adding the protein, wash the filter by adding PBS and centrifuging at high-speed.  

SECTION: Amplification set-up
5. Measure the size of proteins using DLS 
 

SECTION: Amplification set-up
6. Pour one zirconia bead into each well of the   

  

SECTION: Amplification set-up
7. Prepare serial dilutions of sonicated fibrils if needed, make sure to use protein-low-binding tubes and shake the preparation each time using vortex 
SECTION: Amplification set-up
8. Prepare reaction mix containing the monomer mix, PBS and if needed, sonicated fibrils.
SECTION: Amplification set-up
9. Transfer reaction to the plate: start filling the plate with a standard curve samples (seal the portion with a sealing foil)  
Proceed the experimental samples and cover the whole plate with a sealing foil. Spin down the plate  at 500 g for 30 sec. 
SECTION: Amplification set-up
10. Set up the program on a plate reader
 

 

SECTION: Sample collection and analysis
12. Collect replicates at the determined time-points (combine 3 replicates for 1 technical replicate, repeat three times)

SECTION: Sample collection and analysis
13. Centrifuge at 20,000g for 10 min at 10C, collect 90 uL (the pellet should be visible). 
SECTION: Sample collection and analysis
15. Add 4x Laemmli sample buffer to the collected reactions and freeze the samples untill further application 
11. 
SECTION: Sample collection and analysis
14. Transfer the 50 ul of supernatant into a new epperdorf tube, remove soluble fraction from the centrifuged pellets and add 90 ul of fresh PBS
SECTION: Sample collection and analysis
16. Run SDS-PAGE according the protocol described here, use   
 


SECTION: Sample collection and analysis
17. Stop the gel and cover it with 100mL of the fixing solution (50% Methanol, 10% Acetic acid) for 30 min
SECTION: Sample collection and analysis
18. Stain the gel for 1 hr at room temperature with 100 mL of Coomassie stain (0.4g of Coomassie blue   in 200 mL of 40% Methanol)

SECTION: Sample collection and analysis
19. Destain the gel using washing solution consisting of 10% Acetic acid
SECTION: Sample collection and analysis
20. Image the resulting gels using