U plex human α synuclein kit

α-Syn concentration was measured using a U-PLEX Human α-Synuclein Kit (Meso Scale Discovery) according to the manufacturer’s instructions. The measurements were done by users blinded to diagnosis, demographic data, or any other identifying information. Briefly, a biotinylated anti-human α-syn capture antibody was added to small-spot streptavidin-coated wells and incubated at room temperature with shaking at 800 rpm for 1 h. After washing the wells thrice with PBS containing 0.5% (v/v) Tween-20, samples and calibration standards were added along with a Sulfo-TAG-conjugated anti-human α-syn detection antibody and incubated at room temperature with shaking at 800 rpm for 2 h. After washing the wells thrice, read buffer was added and the plates were read using MSD Sector Imager (Model-1250) or QuickPlex SQ 120 instruments. The data were analyzed using Discovery Workbench 4.0 software and quantified with reference to a freshly prepared α-syn standard curve.

Preparation of cell lysates and quantification of total protein concentration were performed using the same procedure as for western blot analysis. α-synuclein protein concentrations were measured with U-PLEX Human α-synuclein Kit (Meso Scale Discovery, Rockville, MD) and MESO SECTOR S 600 (Meso Scale Discovery) as per the manufacturer’s instructions. The biotinylated capture antibody and SULFO-TAG-conjugated detection antibody that we used in the assay were those packaged in the kit. To compare among conditions, quantified α-synuclein concentrations were normalized by total protein concentrations.