This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Rat NFKB-p65. Samples (or Standards) are added to the micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Rat NFKB-p65 and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Rat NFKB-p65, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm. The OD value is proportional to the concentration of Rat NFKB-p65. You can calculate the concentration of Rat NFKB-p65 in the samples by comparing the OD of the samples to the standard curve.