Principle of Assay
Rat NF-kB p65 ELISA Kit (A327108) employs the sandwich enzyme immunoassay technique for the quantitative measurement of rat NF-kB p65 in serum, plasma, and other biological fluids. An antibody specific for NF-kB p65 has been pre-coated onto a 96-well microtiter plate. The standards and test samples are added into the wells and the NF-kB p65 present in each sample is bound to the wells by the immobilized antibody. Following incubation, the wells are washed and then incubated with Biotinylated Anti-NF-kB p65 Antibody, which binds the captured NF-kB p65 present in each well. Following incubation, unbound biotinylated detection antibody is removed by washing, and an HRP-Streptavidin conjugate is added to the wells and the microtiter plate is incubated. Following incubation and washing, substrate solution is then used to visualize the HRP enzymatic reaction by catalysis to produce a blue-coloured product that changes to yellow after addition of acidic stop solution. The density of yellow is proportional to the amount of NF-kB p65 captured in each well. The concentration of NF-kB p65 can then be calculated by reading the O.D. absorbance at 450nm in a microplate reader and referring to the standard curve.
