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 100x to 1x with Assay Diluent. Ensure that there is
enough detection antibody solution for all wells being used 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. Perform 4 consecutive wash steps with gentle shaking between each wash. Prepare the Peroxide and Enhancer
Solution by bringing it to room temperature without exposure to light as these may degrade the substrate. Mix the
Peroxide and Enhancer solution in a 1:1 ratio andstore in room temperature.
8. After the 4th wash step, add 100 μl of the Peroxide/Enhancer solutions into each well and incubate at room
temperature for light development for 5 minutes. The microplate should be kept out of direct light by either covering
with an opaque object or putting it into a dark room. The microplate is now ready to be read by the luminometer.