BetaMark™ x-40 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 1-40 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 appropriate sections below (e.g. sections 2, 3 and 4).
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 1-40 standard to 990uL of standard diluent in intermediate tube #1.
Mix well by inversion, do not vortex.
Remove 10uL from intermediate #1 tube and add to 990uL 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 490uL 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 incubation buffer 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 dilution for 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 to the 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 strip wells if necessary and store in resealable foil pouch at 2-8°C until use.
Add 300uL per well of 1X Wash Buffer (Prepared in step III above).
Dump out wash buffer and pat dry on paper towels.
Add 50uL of each prepared standard to the plate in duplicate or triplicate.
Follow the plate layout outlined inTable 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.
Incubate overnight at 2-8°C.
NOTE: Once diluted with 50uL of diluted HRP detection antibody, the final standard cuve concentrations will be outlined in Table 4.
Please use these concentrations to generate your standard curve.
Remove plate from refrigerator and dump contents.
Wash plate by adding 300uL of 1X Wash Buffer per well.
Dump out 1X Wash Buffer and pat dry.
Repeat steps B-C 4 more times for a total of 5 washes.
Add 200uL TMB subrate to each well.
Read Plate at 620nm.
Go to Biolegend.com for preparation of brain samples for BetaMark™ Beta Amyloid ELISA.
Incubate 40-50 minutes at room temperature in the dark.
Note: If running multiple plates, stagger the addition of substrate so that all plates aren't ready for reading simultaneously.
