Hexanoyl-Lys adduct ELISA Assay

Additional Information

Assay Procedure

1. Prepare Washing solution by mixing one bottle of Washing buffer(x5) and 100 mL of distilled water.
2. Add 50 µL of Standards(A-F) or sample per well. For the Blank well, add 100 µL of Washing solution.
3. Add 50 µL of Primary Antibody to all wells except Blank well. Seal the microtiter plate tightly with Plate Seal. Mix gently by shaking the microtiter plate horizontally. Incubate at 4°C overnight.
4. Reconstitute Secondary Antibody with one bottle of Secondary Antibody Buffer. This is stable for 1 week at 4°C.
5. Remove the plate seal, and pour off the contents of microtiter plate by turning the plate upside down. The use of aspirator is not recommended. Remove the remaining solution by blotting the plate against clean paper towel. Add 250 µL of Washing solution to each well, mix gently by horizontal shaking, and remove the contents similarly. Repeat washing procedure twice and remove the remaining solution of the well.
6. Add 100 µL of Secondary Antibody to all wells. Seal the microtiter plate tightly with Plate Seal. Mix gently by shaking the microtiter plate horizontally. Incubate at room temperature for 1 hour.
7. Prepare Chromogen solution. Add 120 µL of Chromogen to Chromogen. Buffer bottle. Please note that Chromogen solution should be prepared just before use. Alternatively, dilute Chromogen with 100 volumes of Chromogen Buffer.
8. Remove the plate seal, and wash the plate as mentioned at STEP F for 3 times. Remove the remaining solution of the well.
9. Add 100 µL of Chromogen solution to all well, and incubate at room temperature for 15 minutes in the dark.
10. Add 100 µL of Stop Solution to all wells, mix gently, wait for 3 minutes, and measure the absorbance at 450 nm.