Product Description
Specifications
| Size | 1 Plate Kit, 2 Plate Kit, 5 Plate Kit |
| Species | Human |
| Specificity | The olionucleotide/antibody pair provided in this kit recognizes human NF-?B p65 in whole lysates and nuclear extracts. |
| Compatible Sample Types | Cell Lysates, Nuclear Extracts |
| Accession Number | Q04206 |
| Gene Id | 5970 |
| Gene Symbols | RELA, NFKB3 |
| Protein Name / Synonyms | Transcription factor p65 (Nuclear factor NF-kappa-B p65 subunit) (Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3) |
| Quantitative/Semi-Quantitative | Semi-Quantitative |
| Solid Support | 96-well Microplate |
| Design Principle | Sandwich-based |
| Method Of Detection | Colorimetric |
| Research Area | Angiogenesis, Cancer, Inflammation, NFkB Signaling |
| Shipping Type | Dry ice (additional shipping fee) |
| Storage | -20°C |
Introduction
Product Features
- Specific transcription factor-DNA binding assay
- Perfect alternative to EMSA
- Easy to perform in an ELISA format
- Non-radioactive assay
- High throughput (96-well plate format)
- Assay can be completed within 5 hours
Application Notes
- 96-well Strip Microplate pre-coated with DNA probes
- DNA Binding Buffer
- Positive Control Sample
- Specific Competitor DNA probe
- Non-specific Competitor DNA probe
- Assay Reagent
- DTT
- Wash Buffer
- Primary Antibody
- HRP-conjugated Secondary Antibody
- Antibody Diluent Buffer
- TMB One-Step Substrate Reagent
- Stop Solution
- Distilled or deionized water
- 100 ml and 1 liter graduated cylinders
- Tubes to prepare sample dilutions
- Absorbent paper
- Precision pipettes to deliver 2 µl to 1 ml volumes
- Adjustable 1-25 ml pipettes for reagent preparation <lIκBenchtop rocker or shaker
- Microplate reader capable of measuring absorbance at 450 nm
- Prepare all reagents and samples as instructed in the manual.
- Add 100 µl of sample or positive control to each well.
- Incubate 2 h at RT or O/N at 4 °C.
- Add 100 µl of prepared primary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of prepared HRP-secondary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of TMB One-Step Substrate Reagent to each well.
- Incubate 30 min at RT.
- Add 50 µl of Stop Solution to each well.
- Read at 450 nm immediately.
Typical Data
Figure 1
Transcription factor activity assay of NF-κB p65 from nuclear extracts of HeLa cells or HeLa cells treated with TNF-α. After stimulation with TNF-α, activated NF-κB p65 is translocated into the nucleus where it binds with its corresponding DNA.

Figure 2
Transcription factor activity assay of NF-κB p65 from nuclear extracts of HeLa cells or HeLa cells treated with TNF-α with the specific competitor or non-specific competitor. The result shows specific binding of NF-κB p65 to the NF-κB DNA binding site.

Figure 3
Transcription factor activity assay of NF-κB p65 from nuclear extracts of HeLa cells treated with TNF-α and purified NF-κB p50. No cross-reactivity has been observed between NF-κB p65 and p50.

Storage/Stability
Note: The kit can be used within one year if the whole kit is stored at -20°C upon receipt. Avoid repeated freeze-thaw cycles.
citations
Hypoxia reverses the early anti-inflammatory microglial response to the β-amyloid peptide: mitochondrial involvement and beneficial roles of melatonin and naringenin
- Species:
- Human
- Sample Type:
- Other / Unspecified (HMC3 nuclear protein extracts)
- Year:
- 2025
- Journal Name:
- Journal of Translational Medicine
- PubMed ID:
- 40993688
Tumor-associated macrophage (TAM)-secreted CCL22 confers cisplatin resistance of esophageal squamous cell carcinoma (ESCC) cells via regulating the activity of diacylglycerol kinase α (DGKα)/NOX4 axis
- Year:
- 2024
- Journal Name:
- Drug Resistance Updates
- PubMed ID:
- 38387281
Extracellular Vesicles Induce Nuclear Factor-κB Activation and Interleukin-8 Synthesis through miRNA-191-5p Contributing to Inflammatory Processes: Potential Implications in the Pathogenesis of Chronic Obstructive Pulmonary Disease
- Species:
- Human
- Sample Type:
- Other / Unspecified (nuclear extract of 16HBE cells)
- Year:
- 2024
- Journal Name:
- Biomolecules
- PubMed ID:
- 39199417
Immunomodulatory and Antioxidant Properties of a Novel Potential Probiotic Bacillus clausii CSI08
- Species:
- Human
- Sample Type:
- HT-29 cell
- Year:
- 2023
- Journal Name:
- Microorganisms
Focal adhesion kinase (FAK) inhibitordefactinib suppresses the malignant progression of human esophageal squamous cell carcinoma (ESCC) cells via effective blockade of PI3K/AKT axis and downstream molecular network
- Species:
- Human
- Sample Type:
- Conditioned Media (cell culture media)
- Year:
- 2020












