Human NF-KappaB p65 Activity Assay Kit

Cat# TFEH-p65-1

Size : 1PlateKit

Brand : RayBiotech

Contact local distributor :


Phone : +1 850 650 7790

Human NF-KappaB p65 Activity Assay Kit

This assay uses a dsDNA coated plate with canonical NF-kB binding sequences to semi-quantitatively detect active NF-kB in lysates or nuclear extracts. Dry ice shipment (additional fee).
Contact Us for Bulk Request

Product Description

Specifications

Size1 Plate Kit, 2 Plate Kit, 5 Plate Kit
SpeciesHuman
Specificity
The olionucleotide/antibody pair provided in this kit recognizes human NF-?B p65 in whole lysates and nuclear extracts.
Compatible Sample TypesCell 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-QuantitativeSemi-Quantitative
Solid Support
96-well Microplate
Design PrincipleSandwich-based
Method Of DetectionColorimetric
Research AreaAngiogenesis, Cancer, Inflammation, NFkB Signaling
Shipping TypeDry ice (additional shipping fee)
Storage-20°C

Introduction

NF-κB is a transcription factor that plays a pivotal role in a number of physiological cell processes such as growth, apoptosis, and immune response through regulating the expression of a number of cellular genes. The NF-κB family includes five members: cRel, RelB, p65(RelA), p50(p105, precursor of p50), and p52(p100, precursor of p52), which form homo- or heterodimers when in active status. Normally, NF-κB is inactive and retained in the cytoplasm bound to its inhibitory protein IκB. Upon stimulation of various factors such as infection of pathogens including viruses and bacteria or treatment of cytokines such as IL-1 or TNF-α, IκB proteins are phosphorylated, ubiquitinated and finally degraded. Released NF-κB proteins then form homo- or heterodimers, most commonly p50/p65 or p50/p50, which are transported into the nucleus where they bind to specific DNA sequences of targeted genes and activate transcription. NF-κB also induces expression of its own inhibitor, IκBa, which binds to active NF-κB in the nucleus to terminate the induction of gene expression.

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

Kit Components
  • 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
Other Materials Required
  • 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
Protocol Outline
  1. Prepare all reagents and samples as instructed in the manual.
  2. Add 100 µl of sample or positive control to each well.
  3. Incubate 2 h at RT or O/N at 4 °C.
  4. Add 100 µl of prepared primary antibody to each well.
  5. Incubate 1 h at RT.
  6. Add 100 µl of prepared HRP-secondary antibody to each well.
  7. Incubate 1 h at RT.
  8. Add 100 µl of TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 µl of Stop Solution to each well.
  11. 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

Upon receipt, the positive control should be removed and stored at -20° or -80°C. The remainder of the kit can be stored for up to 6 months at 2-8°C from the date of shipment. Opened Microplate Wells or reagents may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack, reseal along entire edge.
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

You might also be interested by the following products:



Cat#
Description
Cond.
Price Bef. VAT
TFEH-p52-1
 1PlateKit 
TFEH-NRF2-1
 1PlateKit 
NE-50
 50extractions