NF-kappaB p65 (Phospho-Ser529)Colorimetric Cell-Based ELISA Kit

From Assay Genie, this cell-based ELISA kit is designed for the accurate measurement of NF-kappaB p65 phosphorylation at Serine 529. The colorimetric assay read at 450 nm is described by the manufacturer as lysate-free, high-throughput, and sensitive, and it detects NF-kappaB p65 protein phosphorylation and expression profile in cells using an indirect ELISA format. Primary antibodies specific for NF-kappaB p65 (Phospho-Ser529) capture the target, while HRP-conjugated secondary antibodies bind the Fc region of the primary antibody, catalysing a colorimetric reaction upon substrate addition.

Phosphorylation of NF-kappaB p65 at Serine 529 is described as a key event in the activation of the NF-kappaB signaling pathway, which regulates genes involved in inflammation, immune response, and cell survival, and whose dysregulation has been linked to cancer, inflammatory conditions, and autoimmune disorders. The assay is reactive with Human, Mouse, and Rat samples, with a dynamic range of > 5000 Cells, and is intended for use with adherent cells. The kit can be used for measuring the relative amounts of phosphorylated NF-kB p65 in cultured cells, as well as for screening the effects that various treatments, inhibitors (e.g. siRNA or chemicals), or activators have on NF-kB p65 phosphorylation.

Two normalization approaches are provided: a monoclonal antibody specific for human GAPDH serves as an internal positive control, and the Crystal Violet whole-cell staining method can be used to determine cell density so that absorbance values can be normalized to cell amounts. The target corresponds to the RELA gene (Gene ID: 5970; UniProt ID: Q04206), with the associated protein reported at a molecular weight of 59,910 Da.