NF-kappaB p100/p52 (Phospho-Ser865)Colorimetric Cell-Based ELISA Kit

From Assay Genie, this Colorimetric Cell-Based ELISA Kit is designed for the precise measurement of NF-kappaB p100/p52 phosphorylated at serine 865 in cell samples. The assay is a convenient, lysate-free, high throughput and sensitive tool that detects NF-kB p100/p52 protein phosphorylation and expression profile in cells, enabling researchers to measure the relative amounts of phosphorylated NF-kB p100/p52 in cultured cells as well as to screen for the effects of various treatments, inhibitors (such as siRNA or chemicals), or activators on NF-kB p100/p52 phosphorylation.

The kit is reactive against Human, Mouse, and Rat samples and employs an indirect ELISA format in which Phospho-Ser865 NF-kappaB p100/p52 is captured by target-specific primary antibodies while HRP-conjugated secondary antibodies catalyze a colorimetric reaction upon substrate addition. Detection is by Colorimetric 450 nm readout, with a stated dynamic range of > 5000 Cells, and the kit is intended for use with Adherent Cells. Due to the qualitative nature of the assay, multiple normalization methods are provided: a monoclonal antibody specific for human GAPDH serves as an internal positive control in normalizing the target absorbance values, and following the colorimetric measurement, the Crystal Violet whole-cell staining method may be used to determine cell density so that absorbance values can be normalized to cell amounts and plating variation adjusted.

The target protein is encoded by the NFKB2 gene (Gene ID: 4791, UniProt ID: Q00653). NF-kappaB p100/p52 is a transcription factor of the nuclear factor-kappaB group that regulates gene expression, immune responses, and inflammation, with phosphorylation at serine 865 known to modulate its activity and influence cell survival, proliferation, and differentiation. The standard procedure seeds 200 µL of 20,000 adherent cells per well of a 96-well plate for overnight incubation at 37 °C, 5% CO2.