Human PPAR-delta Transcription Factor Activity Assay

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

$1,608.00  
TFEH-PPARd-5
+

Antigen Information

Accession Number:
  • Q03181
Gene ID:
  • 5467
Gene Symbols:
  • NR1C2
  • PPARB
  • PPARD
Protein Name & Synonyms:
Peroxisome proliferator-activated receptor delta(PPAR-delta), NUCI, Nuclear hormone receptor 1(NUC1), Nuclear receptor subfamily 1 group C member 2, Peroxisome proliferator-activated receptor beta(PPAR-beta)
Target Species:
  • Human

Assay Format

Specificity:
The olionucleotide/antibody pair provided in this kit recognizes human PPAR-delta 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

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
  • 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 PPAR-delta from purified recombinate PPAR-delta protein with RayBio PPAR-delta TF Activity Assay Kit.

Transcription factor assay of PPAR-delta from purified recombinate PPAR-delta protein with RayBio PPAR-delta TF Activity Assay Kit.

Figure 2
Transcription factor assay of PPAR-delta from 0.3 ug purified recombinate PPAR-delta protein with the specific competitor or non-specific competitor. The result shows specific binding of PPAR-delta to the PPAR-delta DNA binding site.

Transcription factor assay of PPAR-delta from 0.3 ug purified recombinate PPAR-delta protein with the specific competitor or non-specific competitor. The result shows specific binding of PPAR-delta to the PPAR-delta 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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