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  • Transcription factor assay of jun-B from nuclear extracts of K562 cells or K562 cells treated with PMA (50 ng/ml) for 3 hr. A. Western-blot result of jun-B from cytoplasmic and nuclear fractions. B. Transcription factor assay of jun-B from nuclear fractions with the RayBio® Activity Assay Kit.
  • Transcription factor assay of jun-B from nuclear extracts of K562 cells or K562 cells treated with PMA (50 ng/ml) for 3 hr with the specific competitor or non-specific competitor. The result shows specific binding of jun-B to the conserved binding site detected by using the RayBio® jun-B TF Activity Assay Kit.

Human jun-B Transcription Factor Activity Assay

CODE: TFEH-JUNB-1

$398.00

RayBio® Human jun-B Transcription Factor Activity Assay. This assay uses a dsDNA coated plate with canonical jun-B binding sequences to semi-quantitatively detect active jun-B in lysates or nuclear extracts.


Introduction

Activator protein-1 (AP-1) is a sequence-specific transcriptional activator composed of members of the Jun (c-Jun, jun-B, and JunD) and Fos (c-Fos, fosB, Fra1, and Fra2) families in formats of homo- and heterodimers. These proteins belong to the bZIP group of DNA binding proteins with the ability to a common consensus sequence-defined AP-1-binding site. Jun and Fos proteins can also dimerize other basic leucine zipper proteins such as ATF, CCAAT enhancer-binding protein, Maf, and NF-E2. Jun-Jun and Jun-Fos dimers bind preferentially to the TPA responsive element (TRE), whose consensus is TGAGTCA, whereas Jun-ATF dimers prefer to bind to the c-AMP-responsive element (CRE), whose consensus is TGAGCTCA. Inside cells, AP-1 activity is induced by an incredible diversity of signals, including growth factors, cellular stress, ionizing and ultraviolet irradiation, DNA damage, oxidative stress, neuronal depolarization antigen binding by T or B lymphocytes, and cytokines. The mechanisms involved in induction of AP-1 activity are either through changing the expression of AP-1 components or post-translation modification or both to regulate their trans-activity positively or negatively. For example, stimulation by growth factors or by activating mutations in cytoplasmic effectors such as ras and raf, results in AP-1 activation by triggering the ERK signaling pathway. On the other hand, AP-1 responses to proinflammatory cytokines and UV radiation are mostly dependent on two other MAPK cascades, JNK and p38 of MAP kinases. As a result, the AP-1 regulates different target genes executing different biological functions such as cell proliferation, differentiation, apoptosis, or cell death.

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

Specificity

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

Target Protein Information

Synonyms
Transcription factor jun-B
Accession Number
Gene Names
JUNB
Gene ID
3726

Species Detected

Human

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
  • 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
  • Benchtop 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 assay of jun-B from nuclear extracts of K562 cells or K562 cells treated with PMA (50 ng/ml) for 3 hr. A. Western-blot result of jun-B from cytoplasmic and nuclear fractions. B. Transcription factor assay of jun-B from nuclear fractions with the RayBio® Activity Assay Kit.

Figure 2
Transcription factor assay of jun-B from nuclear extracts of K562 cells or K562 cells treated with PMA (50 ng/ml) for 3 hr with the specific competitor or non-specific competitor. The result shows specific binding of jun-B to the conserved binding site detected by using the RayBio® jun-B TF 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.

Product Specifications

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

Array/ELISA Format

  • HIF-1 alpha Signaling Pathway
  • IGF Signaling Pathway
  • p53 Signaling Pathway
  • PI3K Signaling Pathway
  • PKC-Theta Signaling Pathway
  • TGF-beta Signaling Pathway
  • HER/ErbB Signaling Pathway
  • JAK/STAT Signaling Pathway
  • MAPK Signaling Pathway
  • Human
  • 96-well Microplate
  • Sandwich-based
  • Semi-Quantitative
  • Colorimetric
  • Nuclear Extracts
  • Cell Lysates

Note: If you are asking about pricing and you are outside of the US please contact your local RayBiotech distributor.

 

Usage

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

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