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 52 - 53 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 .
