NF-kappaB p65 (Phospho-Ser311)Transcription Factor Activity Assay

The NF-kB p65 Transcription Factor Activity Assay from Assay Genie is a DNA-binding ELISA that quantifies the functional DNA-binding activity of NF-kB p65 phosphorylated at Ser311 in nuclear and cell lysates. Ser311 phosphorylation is a specific post-translational modification that modulates p65 transactivation potential, distinguishing transcriptionally engaged p65 from total protein measured in conventional ELISA formats.

The microplate is pre-coated with an immobilized NF-kB consensus oligonucleotide, capturing only activated p65 dimers capable of sequence-specific DNA binding. A phospho-specific primary antibody selective for the Ser311 epitope detects bound p65, yielding a functional readout of canonical pathway activation across human, mouse, and rat samples.

Researchers apply this assay to characterize NF-kB signaling downstream of TNF-alpha, IL-1beta, LPS, and other pro-inflammatory stimuli in cell culture models. Each microplate kit supports 96 independent determinations; nuclear extracts are prepared in 1.5 mL microcentrifuge tubes using lysis buffer supplemented with protease and phosphatase inhibitors to preserve the Ser311 mark. Store kit components refrigerated at 4 °C, protect from light, and aliquot the phospho-specific antibody to limit freeze-thaw cycles. This reagent is for in vitro research use only and supports pathway screening, inhibitor characterization, and mechanistic investigation of inflammatory and immune responses.