Alpha-Synuclein ELISA Kit Protocol
Notes: Allow kit components to be brought to room temperature before use.
Label a 1L bottle as “1X Wash Buffer”.
Dilute 5X Wash Buffer 1:5 using lab grade water* and mix well.
*Note: Lab grade filtered water such as injection grade, cell culture grade, Reverse Osmosis De-Ionization(RODI).
Label an appropriate sized bottle as “1X Reagent Diluent”.
Dilute 2X Reagent Diluent to 1X by adding 30mL of 2X Reagent Diluent to 30mL of lab grade waterin the bottle labeled “1X Reagent Diluent”.
Mix well by vortex.
Reconstitute lyophilized standard with 50μL of lab grade water and mix well by vortex.
Theconcentration after reconstitution will be 500μg/mL.
Label (2) 1.5mL microcentrifuge tubes as intermediate #1 & intermediate #2.
Aliquot 990μL of 1X Reagent Diluent into intermediate #1 & intermediate #2 tubes.
Remove 10μL from the vial of reconstituted standard and add to 990μL of 1X Reagent Diluent inintermediate tube #1.
Mix well by vortex.
Remove 10μL from intermediate #1 and add to 990μL of 1X Reagent Diluent in intermediate #2tube.
Mix well by vortex.
The final concentration of intermediate tube #2 will be 50ng/mL.
Label (8) 1.5mL microcentrifuge tubes as #1-8.
Add 1280μL of 1X Reagent Diluent to tube #1 and 825μL of 1X Reagent Diluent to tubes #2-8.
Remove 40μL from intermediate #2 and add to 1280μL of 1X Reagent Diluent in tube #1.
Mix well by vortex (this will be the top point of the standard curve, 1500pg/mL).
Remove 550μL from tube #1 and add to 825μL of 1X Reagent Diluent in tube #2.
Mix well by vortex.
Continue making 2.5 fold serial dilutions by adding 550μL of the previous dilution to 825μL of 1XReagent Diluent in tubes #3-7.
Mix well by vortex between each dilution.
Note: Tube #8 will be the zero or blank sample and should only contain 1X Reagent Diluent Dilute samples in 1X Reagent Diluent.
Mix each dilution by vortexing 3 X 2 seconds.
Run samples in duplicate or triplicate.
Note: It is good practice to run at least 2 dilutions for each sample to ensure one of the dilutions fallswithin the linear range of the standard curve.
We recommend a minimum dilution of 1:10.
Note: Dilution factors will vary based on the sample matrix of your experiments.
We recommendrunning a subset of your samples in the kit to determine optimal dilutions and evaluate any matrixeffects.
Failure to do this type of optimization could result in inconclusive data, for which Biolegend isnot responsible.
Remove the plate from the foil pouch.
Add 300μL/well of 1X Wash Buffer.
Dump out wash buffer and pat dry.
Repeat 3 more times for a total of 4 washes.
Add 200μL of each standard to the plate in duplicate or triplicate.
Follow the plate layout below.
Note: The volumes in Table 2 are sufficient for running the standard curve in triplicate.
Add 200μL of each sample dilution to the plate.
Cover the plate with the plate sealer provided.
Incubate overnight at 2-8oC, while shaking.
Notes: Allow kit components to be brought to room temperature before use.
Preparation of Biotinylated Primary Antibody.
Label a 15mL centrifuge tube as “Biotinylated Primary Antibody”.
Dilute the biotinylated primary antibody by adding 6μL* of the Biotinylated Primaryantibody stock to 6mL of 1X Reagent Diluent in the 15mL tube labeled “BiotinylatedPrimary Antibody” 
Note: A quick spin in a centrifuge is suggested prior to pipetting to ensure liquid is at the bottom of the vessel.
Mix well by vortex Remove plate from refrigerator and dump contents.
Add 300μL/well of 1X Wash Buffer.
Dump out wash buffer and pat dry.
Repeat 3 more times for a total of 4 washes.
Add 50μL/well of the Biotinylated Primary Antibody to the plate (prepared above).
Cover and incubate for 2 hours at room temperature.
Label a 1.5mL microcentrifuge tube as “HRP Intermediate”.
Label a 50mL tube as “Diluted Streptavidin HRP”.
Make the “HRP Intermediate” by adding 10μL* of Streptavidin-HRP stock to 990μL of 1X Reagent Diluent in the 1.5mL microcentrifuge tube labeled “HRP Intermediate”.
*Note: A quick spin in a centrifuge is suggested prior to pipetting to ensure liquid is at the bottom of the vessel.
Mix well by vortex.
Remove 150μL from the HRP Intermediate tube and add to 22.35mL of 1X Reagent Diluentin the 50mL tube labeled “Diluted Streptavidin HRP”.
Mix well by vortex.
Remove plate from incubation and dump contents.
Add 300μL/well of 1X Wash Buffer.
Dump out wash buffer and pat dry.
Repeat 3 more times for a total of 4 washes.
Add 200μL/well of Diluted Streptavidin HRP to the plate (prepared above).
Cover and incubate for 1 hour at room temperature.
Remove plate from incubation and dump contents.
Add 300μL/well of 1X Wash Buffer.
Dump out wash buffer and pat dry.
Repeat 3 more times for a total of 4 washes.
Mix chemiluminescent substrates for use:
Add 5.5mL of substrate A to a 15mL centrifuge tube.
Add 5.5mL of substrate B to the same 15mL centrifuge tube.
Mix well by vortex.
Add 100μL of mixed substrate per well.
Note: If reading multiple plates add substrate one plate at a time; do not add substrate to all plates atthe same time.
Add substrate to each plate immediately before reading.
Shake plate on either a plate shaker or using the shaking mechanism within the platereader for 10-15seconds.
Read plate immediately.
Note: The recommended luminometer settings are to read at a mid-range sensitivity level for 1 secondper well.
These settings will vary between plate reader manufacturers, please consult your owner’smanual prior to performing this assay See Biolegend.com for additional data
