How to coat your own plate and run an Invitrogen ELISA kit

Dilute the concentrated coating buffer according to protocol, then use it to dilute the capture antibody to the recommended concentration. Add the antibody solution to the plate wells and incubate overnight at 2-8°C to allow antibody absorption.

Allow wash buffer concentrate to reach room temperature and mix to dissolve any precipitated salts, then dilute in deionized water. Wash the plate by filling wells with wash buffer, soaking, and inverting to decant, repeating according to protocol and tapping dry.

Dilute blocking solution with water and add to the plate wells, then incubate as recommended. After incubation, wash the plate using the same method as previously described.

Reconstitute protein standard vial by gently swirling and inverting five times or briefly vortexing, then let sit for at least ten minutes. Perform a 1:2 serial dilution by adding assay diluent to tubes, then adding reconstituted standard and mixing thoroughly with fresh pipette tips.

Add standards, controls, and samples to the ELISA plate, pre-diluting samples if needed. Dilute biotin conjugate concentrate in assay buffer and add to the plate to allow the biotin-labeled detector antibody to bind antigen at a different site than the capture antibody.

Cover the plate with an adhesive seal and incubate as recommended. After incubation, wash the plate to remove unbound detection antibody. Dilute streptavidin HRP concentrate in assay buffer and add to the plate for enzymatic amplification of the signal.

Wash the plate to remove excess enzyme. Add chromogenic substrate and incubate at room temperature in the dark for 30 minutes, during which the color intensity develops proportionally to the protein amount present.

Add stop solution to halt the enzymatic reaction, turning the solution yellow. Measure the absorbance at 450 nanometers using an appropriate plate reader to obtain your ELISA results.