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
