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Human NRF2 Transcription Factor Activity Assay

RayBio® Human NRF2 Transcription Factor Activity Assay. This assay uses a dsDNA coated plate with canonical NRF2 binding sequences to semi-quantitatively detect active NRF2 in lysates or nuclear extracts. Dry ice shipment (additional fee).

$402.00  
TFEH-NRF2-1
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Antigen Information

Accession Number:
  • Q16236
Gene ID:
  • 4780
Gene Symbols:
  • NFE2L2
  • NRF2
Protein Name & Synonyms:
Nuclear factor erythroid 2-related factor 2 (NF-E2-related factor 2) (NFE2-related factor 2) (HEBP1) (Nuclear factor, erythroid derived 2, like 2)
Target Species:
  • Human

Assay Format

Specificity:
The olionucleotide/antibody pair provided in this kit recognizes human NRF2 in whole lysates and nuclear extracts.
Number of Targets Detected:
1
Species Detected:
  • Human
Compatible Sample Types:
  • Cell Lysates
  • Nuclear Extracts
Design Principle:
  • Sandwich-based
Method of Detection:
Colorimetric
Quantitative/Semi-Quantitative:
  • Semi-Quantitative
Solid Support:
96-well Microplate

Product Specifications

Size:
1, 2, or 5 x 96-Well Strip Microplate Kit

Introduction

Cellular oxidative and electrophilic stress caused by drugs and other xenobiotics, inflammation, and ionizing radiation are associated with an accumulation of reactive oxygen species and electrophilic insults, which contribute to the pathogenesis of various diseases such as cancer, neurodegenerative disease, and atherosclerosis. In order to protect cells from reactive oxygen species and electrophilic insults, the endogenous cellular antioxidant defense system initiates a response to cellular oxidative and electrophilic stress. NRF2 (nuclear factor (erythroid-derived 2)-like 2; NFE2L2) is a key transcriptional factor regulating hundreds of antioxidant and Phase II detoxification genes. Under normal conditions, NRF2 is sequestered in the cytoplasm through binding with Keap1, an actin-binding protein and finally is degraded through the Keap1-dependent ubiquitination. In response to a stimulus, degradation of Keap1 is markedly increased. This leads to the disruption of the Keap1-NRF2 complex and nuclear translocation of NRF2. NRF2 then dimerizes with small Maf proteins and binds to the ARE (antioxidative response element) in promoters of downstream genes to initiate 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
  • 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 NRF2 from nuclear extracts of HepG2 cells or HepG2 cells treated with tBHQ (90uM) for 24 hr. After stimulation activated NRF2 is translocated into the nucleus where it binds with its corresponding DNA. A. Western-blot result of NRF2 from cytoplasmic and nuclear fractions. B. Transcription factor activity assay of NRF2 from nuclear fractions with the RayBio® NRF2 Transcription Factor Activity Assay Kit.

Figure 2
Transcription factor activity assay of NRF2 from nuclear extracts of HepG2 cells or HepG2 cells treated with tBHQ (90uM) for 24 hr with the specific competitor or non-specific competitor. The result shows specific binding of NRF2 to the ARE binding site detected by using the RayBio® NRF2 Transcription Factor Activity Assay Kit.

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.

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Usage

This product is furnished for LABORATORY RESEARCH USE ONLY.
Not for diagnostic or therapeutic use.