Encyclopedia of Experiments: Biological Techniques
A subscription to JoVE is required to view this content. Sign in or start your free trial.
Transcript
To prepare for ELISA, dispense 100 microliters of protein solution into each well of a Polysorp microtiter plate. Close the plates with the lid, and store at 4 degrees Celsius overnight to facilitate the immobilization of protein onto microtiter plate wells. Discard the solution from the microtiter plate by tilting upside down over the sink. Then, wash the wells three times with 300 microliters of PBS per well.
Block the coated wells for 1 hour at room temperature with PBS, containing 2.5 weight volume percent BSA. After removing the blocking solution, wash the wells three times with 300 microliters of PBST per well. Now, add 100 microliters of 50 nanomolar recombinant His-tagged PH to each sample. In the control wells, add only 100 microliters of PBS-BSA. Incubate the plate for 1 hour at room temperature.
Following incubation, discard the protein solution and remove the unbound proteins by washing the wells three times with 300 microliters of PBST per well. Then, add 100 microliters of PBS-BSA, containing horseradish peroxidase-conjugated anti-His polyclonal antibodies.
After incubating for 1 hour at room temperature, wash the wells three times with 300 microliters of PBST per well. Detect the antigen-antibody complexes by adding 100 microliters of ELISA detection reagent to each well. Then, add 50 microliters of 1 molar sulfuric acid per well to stop the reaction. Measure the optical density of the wells at 450 nanometers, using a microplate reader.
