BetaMark™ x-42 ELISA Kit (Colorimetric) Protocol
Label (2) 1.5mL microcentrifuge tubes as intermediate #1 & 2. (use enclosed tubes). 
Add 990uL of standard diluent to intermediate tubes #1 & 2.
Reconstitute one 20ug vial of 1-42 standard with 80uL of Standard Diluent.
Mix well by inversion.
Do not vortex.
Concentration will be 250ug/mL.
Incubate 30 minutes at room temperature.
Note: During 30 minute incubation; proceed thru the next 3 sections. 
After 30 minutes incubation, mix well by inversion, do not vortex.
Once reconstituted, standard must be used within the same day.
Add 10uL from the vial of reconstituted 1-42 standard to 990uL of standard diluent in intermediate tube #1.
Mixwell by inversion, do not vortex.
Remove 10ml from intermediate #1 tube and add to 990mL of standard diluent in intermediate #2 tube.
Mix well by inversion, do not vortex.
The final concentration of standard in intermediate tube #2 will be 25ng/mL.
Label a 50mL centrifuge tube as “1X Incubation Buffer”. 
Dilute 2X Incubation buffer to 1X by adding 10mL of 2X incubation buffer to 10mL of lab grade water* in the 50mL tube labeled “1X Incubation Buffer”.
*Note: Lab grade filtered water such as injection grade, cell culture grade, Reverse Osmosis De-Ionization (RODI).
Mix well by vortexing.
This will be diluent for the standard curve and samples.
Label a 1L container “1X Wash buffer”.
Dilute 5X wash buffer to 1X for use.
Mix 125mL of 5X Wash buffer with 500mL of lab grade water for a total volume of 625mL.
Label (8) 1.5mL microcentrifuge tubes as #1-8 (use enclosed tubes).
Aliquot 240uL of the 1X incubation buffer (made previously in step II) to each of the standard curve tubes (#2-8) and 490mL to tube #1 (standard curve top point).
Remove 10uL from intermediate #2 and add to 490uL 1X incubation buffer in tube #1 (this will be the top point of the standard curve, final concentration will be 250pg/mL).
Mix well by inversion, do not vortex.
Continue making 1.8 fold serial dilutions by adding 300uL of the previous dilution to 240uL of 1X incubationbuffer in tubes #2-7.
Mix well by inversion between each dilution.
Dilute samples in 1X incubation buffer to 2X concentration.
Mix well by inversion.
For example, if the final sample dilution should be 1:10, dilute the sample 1:5 in 1X incubation buffer.
The sample will then be diluted 1:2 in step VII for a final dilution factor of 1:10.
Run samples in duplicate or triplicate.
Note: All sample matrices will perform differently in the kit.
It is important that you determine the ideal dilutionfor your particular sample type.
It is good practice to run 2-3 dilutions per sample to ensure at least 1 dilution falls within the range of the standard curve.
For most sample types, a good starting dilution would be 1:5-1:10.
Due tothe format of the assay, samples are not able to run neat.
Label a 15mL tube as “Diluted HRP Detection Antibody”. 
Add 6mL of 1X Incubation buffer to the tube labeled “Diluted HRP Detection Antibody”. 
Add 6uL of HRP Detection Antibody and mix well by vortexing. 
Remove plate from foil pouch.
Remove extra stripwells if necessary and store in resealable foil pouch at 2-8°C until use.
Add 300uL per well of 1X Wash Buffer.
Note: Use of automated plate washer is highly recommended. 
Dump out wash buffer and pat dry on paper towels.
Add 50uL of each standard to the plate in duplicate or triplicate.
Follow the plate layout outlined in Table 3 below.
Note: Wells E1-E3 contain the zero or blank sample (Std #8). 
Add 50uL of each sample to the plate in duplicate or triplicate.
Add 50uL per well of diluted HRP detection antibody to all wells.
Cover plate with plate sealer.
Mix the plate gently on a plate shaker for one minute.
Incubate overnight at 2-8°C.
NOTE: Once diluted with 50uL of diluted HRP detection antibody, the final standard cuve concentrations will beoutlined in Table 4.
Please use these concentrations to generate your standard curve.
Remove plate from refrigerator and dump contents if washing manually.
Wash plate 5X by adding 300uL 1X wash buffer per well.
Note: Use of automated plate washer is highly recommended Mix chemiluminescent substrates for use: 
i. Add 5.5mL of substrate A to a 50mL centrifuge tube.
ii. Add 5.5mL of substrate B to the same 50mL centrifuge tube.
Mix well by vortexing 3 X 2 seconds. 
Add 100uL of mixed substrate per well.
Notes: We recommend pouring substrate into a reservoir and use a multi-channel pipette to dispense.
If reading multiple plates add substrate one plate at a time, do not add substrate to all plates at the same time.
Add substrate to each plate immediately before reading.
Shake plate on either a plate shaker or using the shaking mechanism within the luminometer for 15 seconds.
Read plate immediately.
Plates must be read within 5 minutes of adding substrate.
Notes: 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 Preparation of Brain Samples for BetaMark™ Beta Amyloid ELISA
