NF-kappaB p65 Transcription Factor Activity Assay

The NF-kappaB p65 Transcription Factor Activity Assay from Assay Genie is a DNA-binding ELISA that quantifies the DNA-binding activity of the p65 (RelA) subunit of the NF-κB transcription factor family. Unlike antibody-capture immunoassays that quantify total protein levels, this kit detects only the active fraction of p65 capable of binding the κB consensus sequence, delivering a functional readout of canonical NF-κB pathway activation rather than a simple abundance measurement.

Each microplate well is pre-coated with a double-stranded oligonucleotide probe harboring the NF-κB consensus binding site. Activated p65 present in nuclear extracts engages this probe and is detected with a p65-specific primary antibody followed by an HRP-conjugated secondary reagent. The kit accommodates nuclear and cell lysates prepared from human, mouse, and rat samples, supporting studies of inflammation, immune signaling, apoptosis, oxidative stress, and oncogenic signaling cascades. A typical loading of 10 µg nuclear extract per well is recommended.

This format provides a non-radioactive, higher-throughput alternative to electrophoretic mobility shift assays (EMSA), simplifying screening of pharmacological compounds, cytokines, siRNAs, and pathway inhibitors that modulate NF-κB activity. Reagents should be stored refrigerated and the nuclear extract aliquoted to limit freeze-thaw cycles, which erode transcription factor binding activity.