Abcam’s NFκB p65 (pS536) in vitro SimpleStep ELISA™ (Enzyme-Linked Immunosorbent Assay) kit is designed for the semi-quantitative measurement of NFκB p65 (pS536) protein in Human and mouse cells.

The SimpleStep ELISA™ employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.

As of October 2019, this kit was reformulated with new antibodies to maintain continued long term supply.