ELISA-Based Reporter Assay to Study the Receptor-Ligand Interaction in BW Cells
Transcription
Use 50,000 transfected BW cells with their desired targets in a single well of a 96-well plate. Incubate the plate for 48 hours. Then, freeze the plate at -20 degrees Celsius.
Coat an ELISA plate with anti-Mouse IL-2 antibody. Place 0.05% micrograms of anti-mouse-IL-2 in a volume of 50 microliters of PBS in each well. Incubate the coated plate overnight at 4 degrees Celsius.
Use a brief incubation period at 37 degrees Celsius to thaw the frozen BW cells that were previously incubated with their targets. Then, centrifuge the plate, and transfer 100 microliters of the supernatant from each well to the pre-coated ELISA plate, and incubate at 37 degrees Celsius for 2 hours.
After this, add biotin anti-mouse-IL-2 to the ELISA plate. Then, after incubation and washing, add HRP-conjugated Streptavidin to the plate. After incubation and washing, add 100 microliters of the TMB substrate solution to each well of the ELISA plate. Finally, read the ELISA plate at 650 nanometers.
