1. Dilute Protein Standard with Assay Diluent within the range of the Protein Standard in a series of microfuge tubes.
Mix each tube thoroughly by inverting several times or by vortexing lightly to ensure proper equilibration. Add 100 μl of
each serial dilution step into the wells of a specified row or column of the 96-well microtiter plate in duplicate or
triplicate and incubate at room temperature for 2 hours. Unknown Samples of Interest can be serial diluted with Assay
Diluent to concentrations within the detection range of this assay kit and added to the plate at 100 μl per well.
Blank Control is defined as 100 ul of Assay Diluent per well. Seal the microplate air-tight using parafilm if readily available.
2. Aspirate the protein standard solution out of the microplate wells. If your lab does not have a vacuum-based aspirator,
you may dump the solutions from the microplate into a waste container and blot 3-4 times on a stack of paper towels
until most or all of the liquid is removed from the wells. Dilute the 15x Wash Buffer to 1x using pure H2O. Add 300-400 μl
of 1x Wash Buffer to each well being used and gently shake for 5-7 minutes on an orbital shaker. Perform this wash step
4 times consecutively.
3. After the 4th wash step, dilute the detection antibody solution 1:200 in Assay Diluent to 0.42ug/ml. Mix the test tube
either by inverting several times or vortexing to ensure proper equilibration. Ensure that there is enough detection
antibody solution for all wells being used. Add 100 μl of the diluted detection antibody solution into each well, seal the
plate and incubate at room temperature for 2 hours.
4. Remove the detection antibody solution out of the microplate wells by either vacuum-based aspirator or paper towel
blotting. Perform 4 consecutive wash steps with gentle shaking between each wash.
5. Dilute the 400x Streptavidin-HRP by 1:400 using Assay Diluent to a 1x Streptavidin-HRP solution.
6. After the 4th wash step, add 100 μl of 1x Streptavidin-HRP solution into each well and incubate at room temperature
for 30 minutes.
7. Remove the 1x Streptavidin-HRP solution out of the microplate wells by either vacuum-based aspirator or paper towel
blotting. Prepare the Ready-to-Use Substrate by bringing it to room temperature without exposure to fluorescent or UV
light as these may degrade the substrate. Perform 4 consecutive wash steps with gentle shaking between each wash.
8. After the 4th wash step, add 100 μl of Ready-to-Use Substrate solution into each well and incubate at room
temperature for approximately 10-15 mins. The microplate should be kept out of direct light by either covering with an
opaque object or putting it into a dark room. Closely monitor the color development as some wells may turn blue very
quickly depending on analyte and/or detection antibody-HRP concentrations. Once the blue color has ceased to develop
further, immediately add 100 μl of Stop Solution to each well being used. The color in the wells should immediately
change from blue to yellow.
9. The microplate is now ready to be read by a microplate reader. Within 30 minutes of adding the Stop Solution,
determine the optical density (absorbance) of each well by reading the plate with the microplate reader set to 450 nm.
If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings
at 540 nm or 570 nm from the readings at 450 nm.