Recombinant a-synuclein pre-formed fibril generation

Reconstitute 1mg of lyophilized monomeric synuclein with 100µL cold sterile DPBS On ice (do not pipet, close tube immediately).

Transfer tubes on wet ice to 4°C cold room and rotate on tube rotator for 0h 10m 0s.

Centrifuge for 0h 10m 0s at 15000x g,0h 0m 0s at 4°C.

Transfer supernatant to clean Protein-LoBind tube.

Prepare 5µL aliquots of serial dilutions (1:10, 1:25, 1:50, 1:100) and measure concentration with Nanodrop (A280 - MW=14,5 kDa; Extinction coefficient ε for human α-syn = 5,960 M^-1cm^-1).

Dilute down to final concentration of 5mg/mL, use 100µL (100µL-500µL) aliquots for reproducible results.

Take 1µL-2µL aliquot of monomer and dilute to 1ul/ml for electron microscopy, flash-freeze in dry ice – ethanol slurry.

Seal tubes with parafilm if no tube lock.

Place the tube in an orbital thermomixer with a heated lid for 168h 0m 0s at 37°C, shaking at 1000rpm,0h 0m 0s.

Gently flick the tube to resuspend a-syn fibrils.

a. Fibrils for quality control steps can be stored at 37Room temperature``0h 10m 0s.

Prepare aliquots for QC assays:

a. 5µL – thioflavin T binding assay.

b. 2µL for TEM.

For long-term storage (12-18 months), flash-freeze fibrils in liquid nitrogen and store at -80°C.