Overview

  • Product name

    PPAR gamma Transcription Factor Assay Kit
  • Detection method

    Colorimetric
  • Sample type

    Adherent cells, Suspension cells, Nuclear Extracts
  • Assay type

    Semi-quantitative
  • Species reactivity

    Reacts with: Mouse, Rat, Human
    Predicted to work with: Mammals
  • Product overview

    PPAR gamma Transcription Factor Assay kit ab133101 is a non-radioactive, sensitive method for detecting specific transcription factor DNA binding activity in nuclear extracts.


    A 96 well enzyme-linked immunosorbent assay (ELISA) replaces the cumbersome radioactive electrophoretic mobility shift assay (EMSA). A specific double stranded DNA (dsDNA)sequence containing the peroxisome proliferator response element (PPRE) is immobilized onto the bottom of wells of a 96 well plate. PPARs contained in a nuclear extract bind specifically to the PPRE. PPAR gamma is detected by addition of specific primary antibody directed against PPAR gamma. A secondary antibody conjugated to HRP is added to provide a sensitive colorometric readout at 450 nm. PPAR gamma will not crossreact with PPAR delta or PPAR alpha.

  • Notes

    Peroxisome proliferator-activated receptors (PPARs) are ligand activated nuclear receptors. Three PPAR subtypes have been identified: alpha, delta and gamma. PPARs can be activated by polyunsaturated fatty acids, eicosanoids and various synthtic ligands.

    PPAR gamma is primarily expressed in adipose tissue and to a lesser extent in the colon, immune system and the retina. PPAR gamma was first identified as regulator of adipogenesis, but also plays an important role in cellular differentiation, insulin sensitization , atherosclerosis and cancer.

  • Platform

    Microplate reader

Properties

Images

  • 3T3-L1 cells were differentiated to adipocytes with 1 uM dexamethasone (ab120743), 1 ug x mL-1 insulin (ab123768) and 0.5 mM IBMX (ab120840). From the third day, the cells were grown in normal medium with the addition of only 1 ug x mL-1 insulin. From day six, the cells were cultured in normal medium for an additional two days. 40 uL of nuclear extracts (ab113474) were tested in duplicates (+/- SD).

  • Different volumes of positive control with inhibitor (duplicates, +/-SD).

  • Different volumes of positive control (duplicates, +/-SD).

  • Panel A: Increasing amounts of positive control (total lysate) are assayed for PPAR gamma DNA-binding activity using ab133101.

    Panel B: PPAR gamma DNA-binding assays are performed in the presence of competitive dsDNA. The decrease in signal caused by addition of competitive dsDNA confirms the assay specificity.

Protocols

References

This product has been referenced in:

  • Shou X  et al. Emodin, A Chinese Herbal Medicine, Inhibits Reoxygenation-Induced Injury in Cultured Human Aortic Endothelial Cells by Regulating the Peroxisome Proliferator-Activated Receptor-? (PPAR-?) and Endothelial Nitric Oxide Synthase (eNOS) Signaling Pathway. Med Sci Monit 24:643-651 (2018). Functional Studies ; Human . Read more (PubMed: 29386501) »
  • Hsu CP  et al. Endothelial-cell inflammation and damage by reactive oxygen species are prevented by propofol via ABCA1-mediated cholesterol efflux. Int J Med Sci 15:978-985 (2018). Read more (PubMed: 30013438) »
See all 10 Publications for this product

Customer reviews and Q&As

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ab133101 - PPARy transcription factor assay kit.

This product was used to detect activated PPARy in swine brain that had been subjected to hemorrhagic shock and TBI and treated with normal saline (NS), Hextend (HEX), or Hextend + valproic acid (HEX + VPA). 40mg of brain per sample was homogenized. Nuclear extraction was performed using an Abcam nuclear extraction kit. Protein levels were balanced. The ELISA was performed per the manufacturer's instructions. Samples were incubated overnight at 4 degrees. Colorimetric reaction was brisk and was monitored for progression (per instructions, at 650 nm). Stop solution was added after 30 minutes of agitation at room temperature (control absorbance = 0.5-0.6). The use of competitive dsDNA produced an expected decrease in absorbance (0.3-0.4). All samples were run in duplicates. The average absorbance for each group was as follows:

SHAM 0.55
NS 0.82
HEX 0.76
HEX + VPA 0.73

These findings were not statistically significant and did not support our hypothesis. As such, they will not be published.

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Submitted Nov 23 2015

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