Introduction
Activator protein-1 (AP-1) is a sequence-specific transcriptional activator composed of members of the Jun (c-Jun, JunB, 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 bind 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 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 the induction of AP-1 activity are mediated through changes in the expression of AP-1 components or post-translational modification or both, resulting in regulation of 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 mostly depend on two other MAPK cascades, JNK and p38. The result is that AP-1 regulates various target genes executing 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
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
- 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 c-JUN from nuclear extracts of K562 cells or K562 cells treated with PMA (50 ng/ml) for 3 hr. A. Western blot of c-Jun from cytoplasmic and nuclear fractions. B. Detection of c-Jun from nuclear fractions with the RayBio® Activity Assay Kit.
Figure 2
Transcription factor assay of c-Jun 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 c-Jun to the conserved binding site detected by using the RayBio® c-Jun 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.
