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
