Human NF-κB RelB Transcription Factor Activity Assay

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

CODE: TFEH-RELB-1

Number of Plates:

Antigen Information

Accession Number:

Q01201

Gene ID:

5971

Gene Symbols:

RELB

Protein Name & Synonyms:

Transcription factor RelB (I-Rel)

Target Species:

Human

Target Name:

RelB

Assay Format

Specificity:

The olionucleotide/antibody pair provided in this kit recognizes human SOX2 in whole lysates and nuclear extracts.

Number of Targets Detected:

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-Ã± (IKKÃ±) is activated and triggers p100 phosphorylation and processing, which generates RelB and results in RelB/RelB 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 value.

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
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Microplate reader capable of measuring absorbance at 450 nm

Protocol Outline

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

Human NF-κB RelB Transcription Factor Activity Assay(图1)

Figure 2
Transcription factor assay of NF-κB RelB 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 RelB to the NF-κB non-canonical DNA binding site.

Human NF-κB RelB Transcription Factor Activity Assay(图2)

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.