This NF-kB p65 (phospho Ser529) Cell Based ELISA Kit allows for the detection of NF-kB p65 (phospho Ser529) and the effects that certain stimulation conditions have on NF-kB p65 (phospho Ser529) expression in different cell lines. Qualitative determination of NF-kB p65 (phospho Ser529) concentration is achieved by an indirect ELISA format. In essence, NF-kB p65 (phospho Ser529) is captured by Anti-NF-kB p65 (phospho Ser529) Antibodies while Dye 1-conjugated and Dye 2-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the dye conjugated to the secondary antibody can emit light at a certain wavelength given proper excitation, hence allowing for a fluorometric detection method. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target RFU values. Anti-NF-kB p65 Antibody is also included for normalization purposes. The RFU values obtained for the non-phosphorylated target can be used to normalize the RFU value for the phosphorylated target.