Bring all reagents to room temperature before use. If crystals have formed in buffer solution, warm to room temperature and mix gently until the crystals have completely dissolved.
Bring all reagents and samples to room temperature before use. It is recommended that all samples and standards be assayed in duplicate.
1. Prepare all reagents, working standards, and samples as directed in the previous sections.
2. Remove unused microplate strips from the plate frame, return them to the foil pouchcontaining the desiccant pack, and reseal.
3. Wash each well three times with Wash Buffer (300 μL/well) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. Remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
4. Add 100 μL of each serially diluted protein standard or test sample per well including a zero standard. Ensure reagent addition is uninterrupted and completed within 15 minutes. Cover/seal the plate and incubate for 2 hours at room temperature.
5. Repeat the aspiration/wash as in Step 3.
6. Add 100 μL of Detection Antibody in working concentration to each well. Cover/seal the plate and incubate for 1 hour at room temperature.
7. Repeat the aspiration/wash as in Step 3.
8. Add 200 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Protect from light.
9. Add 50 μL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 20 minutes, using a microplate reader set to 450 nm.
Calculation of Results
If samples generate values higher than the highest standard, dilute the samples and repeat the assay. Calculate the mean absorbance for each standard, control and sample and subtract average zero standard optical density (O.D.)
Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. Most graphing software can help make the curve and a four parameter logistic (4-PL) usually provide the best fit, though other equations (e.g. linear, log/log) can also be tried to see which provides the most accurate. Extrapolate the target protein concentrations for unknown samples from the standard curve plotted.