Human NF-κB p52 Transcription Factor Activity Assay

RayBio® Human NF-κB p52 Transcription Factor Activity Assay. This assay uses a dsDNA coated plate with canonical NF-κB p52 binding sequences to semi-quantitatively detect active NF-κB p52 in lysates or nuclear extracts. Dry ice shipment (additional fee).

$402.00  
TFEH-p52-1
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Antigen Information

Accession Number:
  • Q00653
Gene ID:
  • 4791
Gene Symbols:
  • LYT10
  • NFKB2
Protein Name & Synonyms:
Nuclear factor NF-kappa-B p100 subunit (DNA-binding factor KBF2) (H2TF1) (Lymphocyte translocation chromosome 10 protein) (Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2) (Oncogene Lyt-10) (Lyt10) [Cleaved into: Nuclear factor NF-kappa-B p52 subunit]
Target Species:
  • Human
Target Name:
NF-kB P52

Assay Format

Specificity:
The olionucleotide/antibody pair provided in this kit recognizes human SOX2 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

The non-canonical NF-κB pathway is an important arm of NF-κB signaling that predominantly functions through activation of the p52/RelB NF-κB complex. This pathway regulates important biological functions, such as lymphoid organogenesis, B-cell survival and maturation, dendritic cell activation, and bone metabolism. Moreover, deregulated non-canonical NF-κB signaling is associated with lymphoid malignancies. In contrast to the canonical NF-κB pathway, in which NF-κB is inactive and retained in the cytoplasm by its inhibitor, IκB, and only transported to the nucleus through modification and degradation of IκB upon stimulation of various factors, the non-canonical pathway depends on the inducible processing of p100, that functions as both the precursor of p52 and a RelB-specific inhibitor. When cells are stimulated by of a subset of TNF receptor family members, the downstream kinase IκB kinase-α (IKKα) is activated and triggers p100 phosphorylation and processing, which generates p52 and results in RelB/p52 NF-κB complex translocation to the nucleus where it regulates the corresponding downstream genes. It is increasingly clear that this pathway of NF-κB activation differs significantly from the canonical NF-κB pathway in its signaling mechanisms. Therefore, better understanding of the mechanism regulating non-canonical NF-κB activation has important therapeutic values.

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 assay of NF-κB p52 from nuclear extracts of HL60 cells or HeLa cells with the RayBio®NF-κBp52 TF Activity Assay.

Transcription factor assay of NF-κB p52 from nuclear extracts of HL60 cells or HeLa cells with the RayBio NF-κBp52 TF Activity Assay.

Figure 2
Transcription factor assay of NF-κB p52 from nuclear extracts of HL60 cells or HeLa cells with the specific competitor or non-specific competitor. The result shows specific binding of NF-κB p52 to the NF-κB non-canonical DNA binding site.

Transcription factor assay of NF-κB p52 from nuclear extracts of HL60 cells or HeLa cells with the specific competitor or non-specific competitor. The result shows specific binding of NF-κB p52 to the NF-κB non-canonical DNA binding site.

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.

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