Thaw down a-syn monomer and/or sonicated fibril aliquots. Use water-bath.

Do not generate bubbles by pipetting or shaking

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

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

Use

to filter the diluted monomer concentration. Prior to adding the protein, wash the filter by adding PBS and centrifuging at high-speed.

Measure the size of proteins using DLS

Dynamic Light Scattering measurements

Pour one zirconia bead into each well of the

Prepare serial dilutions of sonicated fibrils if needed, make sure to use protein-low-binding tubes and shake the preparation each time using vortex

Prepare reaction mix containing the monomer mix, PBS and if needed, sonicated fibrils.

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.

Set up the program on a plate reader

Equipment

Value	Label
Omega - RT-QuIC / PRION Version - Fluorescence Base	NAME
BMG Fluostar plate reader	TYPE
BMG	BRAND
0415-102P	SKU
http://www.bmglabtech.com/clariostar-plus/?utm_term=clariostar&utm_campaign=usa.roi.microplatereaders+products&utm_source=adwords&utm_medium&gclid=Cj0KCQjw4ImEBhDFARIsAGOTMj9ZLSjhHBhKWAli17cxj6V54U96GSg3qsfuFrfP1cETTLgLbn-kdUIaAkovEALw_wcB	LINK

Collect replicates at the determined time-points (combine 3 replicates for 1 technical replicate, repeat three times)

Centrifuge at 20,000g for 10 min at 10C, collect 90 uL (the pellet should be visible).

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

Add 4x Laemmli sample buffer to the collected reactions and freeze the samples untill further application

Run SDS-PAGE according the protocol described here, use

Immunoblotting of α-synuclein species

Stop the gel and cover it with 100mL of the fixing solution (50% Methanol, 10% Acetic acid) for 30 min

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)

Destain the gel using washing solution consisting of 10% Acetic acid

Image the resulting gels using

Equipment

Value	Label
ChemiDoc™ MP Imaging System	NAME
Imaging System	TYPE
Bio-rad	BRAND
12003154	SKU
http://www.bio-rad.com/	LINK