NF-kappaB p65 (Phospho-Thr254)Colorimetric Cell-Based ELISA Kit

This In-Cell ELISA kit from Assay Genie is designed for the detection of phosphorylated NF-κB p65 at threonine 254 in cell samples. It provides a convenient, lysate-free, high-throughput assay that can detect NF-κB p65 protein phosphorylation and expression profile in cultured cells, enabling researchers to measure the relative amounts of phosphorylated NF-κB p65 and to screen for the effects of various treatments, inhibitors such as siRNA or chemicals, or activators on NF-κB p65 phosphorylation. The assay is intended for use with adherent cell samples.

NF-κB p65 is a key transcription factor involved in regulating the expression of genes related to inflammation, immune response, and cell survival. Phosphorylation at threonine 254 plays a crucial role in its activation and translocation to the nucleus, where it exerts its transcriptional activity. The kit is therefore valuable for studying the activation status of NF-κB p65 and its role in cellular processes including inflammation, cancer, and autoimmune diseases.

The assay uses an indirect ELISA format in which NF-κB p65 (Phospho-Thr254) is captured by phospho-specific primary antibodies, and HRP-conjugated secondary antibodies bind the Fc region of the primary antibody to catalyze a colorimetric reaction upon substrate addition. A monoclonal antibody specific for human GAPDH is included as an internal positive control for normalization, and a Crystal Violet whole-cell staining method is used to determine cell density. The kit is reactive with human, mouse, and rat samples, uses colorimetric detection at 450 nm, and has a dynamic range of > 5000 cells. The target corresponds to the RELA gene (Gene ID: 5970, UniProt ID: Q04206).