Direct ELISA

Dilute 100ng - 500ng of protein of interest (e.g. α-synuclein) in Takeda buffer per well and add 30µL total liquid per well to 384-well Nunc Maxisorp plate.

Seal with removable clear adhesive cover.

Centrifuge the plate at 1000x g,0h 0m 0s for 0h 1m 0s to pull down protein onto the plate. Leave for 4h 0m 0s at37°C or at4°C .

Use the plate washer with 5x with 100µL PBST.

Block with 100µL Blockace per well. Fill wells from the bottom, being sure to avoid

leaving any bubbles in the wells.

Seal with a removable clear adhesive cover and leave for 4h 0m 0s at 37°C or at 4°C.

Use the plate washer with 5x with 100µL PBST.

Use C buffer to dilute reporter antibody. Vortex immediately before pipetting.

Using multichannel, fill 91, dispense 30µL three times.

Seal with removable clear adhesive cover and centrifuge plate at 1000x g,0h 0m 0s for 0h 1m 0s.

Incubate for 4h 0m 0s at 37°C or at 4°C.

Use C buffer to dilute HRP-conjugated secondary reporter antibody.

Add 30µL per well. For goat-anti-mouse/rabbit use at 1:5-20K.

Seal with removable clear adhesive cover and centrifuge plate at 1000x g,0h 0m 0s for 0h 1m 0s.

Incubate for 1h 0m 0s at 37°C.

Use the plate washer with 5x with 100µL PBST.

Add 30µL TMB reagent per well.

Develop for 10 - 30 min.

Quench using 30µL 10% phosphoric acid per well.

Read plate on the Spectramax or similar plate reader. 384-495 nm for unquenched

reactions, 450 nm for quenched reactions.