Introduction
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear hormone receptor superfamily. It includes three subtypes PPAR-alpha, PPAR-gamma, and PPAR-delta. Each one mediates the physiological actions of a large variety of fatty acids (FAs) and FA-derived molecules. The PPARs contain the canonical domain structure common to other nuclear receptor family members, including the amino-terminal AF-1 trans activation domain, a DNA-binding domain, and a dimerization and ligand-binding domain with a ligand-dependent trans activation function AF-2 at the carboxy-terminal region. PPAR-alpha, PPAR-delta, and PPAR-gamma play an essential role in energy metabolism, however, they differ in the spectrum of their activity. PPAR-alpha is highly expressed in hepatocytes, enterocytes, vascular and immune cell types. PPAR-alpha plays a central role in lipid and lipoprotein metabolism, and thereby decreases dyslipidemia associated with metabolic syndrome. PPAR-gamma has been known to regulate adipocyte differentiation, FA storage and glucose metabolism, and is a target of antidIκBetic drugs. PPAR-gamma also enhances the expression of a number of genes involved in glucose and lipid metabolism. PPAR-delta is expressed almost ubiquitously with the highest level of expression found in colon, small intestine, liver and keratinocytes. It is a general regulator of fatty acid oxidation in many tissues, where it promotes FA metabolism and suppresses macrophage derived inflammation. The PPARs form heterodimers with the RXRs in cellular nucleus and can regulate gene expression through binding either PPAR ligands or RXR ligands. The formed heterodimers bind to PPAR-responsive elements (PPREs) that consist of direct repeats (DRs) with the core sequence AGG(A/T)CA separated by one or two base-pairs, designated DR1 and DR2, respectively. In the absence of ligand, the heterodimer retains in the nucleus binding to PPREs in a complex with transcriptional co-repressors. Upon ligand binding to PPARs or RXR, the complex makes conformational transfer that facilitates PPARs/RXR heterodimers binding with co-activator from co-repressors. The activated complex therefore starts transcriptional activation of target genes.
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 PPAR-gamma from purified recombinate PPAR-gamma protein with the RayBio PPAR-gamma TF Activity Assay Kit.
Figure 2
Transcription factor assay of PPAR-gamma from 0.3 ug purified recombinate PPAR-gamma protein with the specific competitor or non-specific competitor. The result shows specific binding of PPAR-gamma to the PPAR-gamma 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.
