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Pparγ transcription factor assay kit

Manufactured by Abcam
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The PPARγ transcription factor assay kit is a laboratory tool designed to detect and quantify the activity of the PPARγ (Peroxisome Proliferator-Activated Receptor Gamma) transcription factor in cellular extracts. It provides a simple and efficient method to measure PPARγ DNA-binding activity.

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Lab products found in correlation

Nuclear extraction kit, by Abcam (5 mentions) Nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions) Anti cyclophilin d, by Abcam (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Synergy ht, by Agilent Technologies (1 mentions) Infinite m200, by Tecan (1 mentions) Mouse il6 elisa kit, by Biorbyt (1 mentions) Mouse tnfalpha elisa kit, by Biorbyt (1 mentions) Mouse leptin elisa kit, by Abcam (1 mentions) Mouse adiponectin elisa kit, by Abcam (1 mentions) Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions) Qubit protein assay kit, by Thermo Fisher Scientific (1 mentions) Spectramax m2, by Molecular Devices (1 mentions) Rapamycin, by Merck Group (1 mentions) Cellular senescence assay kit, by Merck Group (1 mentions) Gw9662, by Merck Group (1 mentions)

15 protocols using pparγ transcription factor assay kit

1

Mitochondrial Stress Response Analysis

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Tetramethylrhodamine methyl
ester (TMRM), Thiol Tracker Violet, 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein
diacetate (carboxy-H2DCFDA), MitoProbe Transition Pore
Assay Kit, Pierce Protease Inhibitor Mini Tablets, Pierce Phosphatase
Inhibitor Mini Tablets, phosphate buffered saline (PBS) (pH 7.2),
Supersignal West Pico Luminiscent Substrate, Supersignal West Femto
Maximum Sensitivity Substrate, oligo-dT primers, RevertAid Reverse
Transcriptase, and PowerUp SYBR Green Master Mix were purchased from
Thermo Fisher Scientific. RSG, CsA, Nuclear Extraction Kit, PPARγ
Transcription Factor Assay Kit, and anti-cyclophilin D (ref ab110324,
lot GR3373678-3) and anti-lamin B1 (ref ab16048, lot GR3244890-1)
antibodies were obtained from Abcam. Anti-PPARγ (ref MAB3827,
lot 2470389) and anti-β-actin (ref MAB1501, lot 3800739) antibodies,
HDAC3 Activity Assay Kit, PVDF membrane, and other reagent grade chemicals
were purchased from Merck. Locke’s buffer was composed of 154
mM NaCl, 5.6 mM KCl, 1.3 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 10 mM HEPES. Compounds were dissolved in DMSO,
and serial dilutions were done in cell medium. Vehicle concentration
was always kept under 0.5% in cell treatments. Control cells were
treated with the higher DMSO concentration used in each assay to test
the vehicle effect.
Alvariño R., Alfonso A., Tabudravu J.N., González-Jartín J., Al Maqbali K.S., Elhariry M., Vieytes M.R, & Botana L.M. (2024). Psammaplin A and Its Analogs Attenuate Oxidative Stress in Neuronal Cells through Peroxisome Proliferator-Activated Receptor γ Activation. Journal of Natural Products, 87(4), 1187-1196.
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2

Measuring PPARγ Transcriptional Activity

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We detected the PPARγ activity by using the PPARγ Transcription Factor Assay Kit (ab133101, abcam, MA, USA) according to the manufacturer's protocol. Briefly, he nuclear protein extraction was collected as described earlier and used for the determination of PPARγ activity activity by spectrophotometer reader with absorbance at OD 450 nm. All measured values were detected by Synergy HT (BioTek, VT, USA).
Hsu C.P., Lin C.H, & Kuo C.Y. (2018). Endothelial-cell inflammation and damage by reactive oxygen species are prevented by propofol via ABCA1-mediated cholesterol efflux. International Journal of Medical Sciences, 15(10), 978-985.
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3

PPARγ Transcriptional Activity Assay

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The DNA binding activity of PPARγ was assessed using a PPARγ Transcription Factor Assay Kit (Abcam), as described previously [21 ]. Briefly, the nuclear proteins were incubated in wells immobilized with specific PPAR-binding element (PPRE) sequences with the primary anti-PPARγ antibody, HRP-conjugated secondary antibody subsequently, and the absorbance was measured at 450 nm to determine the transcriptional activity of PPARγ.
Kim J.T., Napier D.L., Kim J., Li C., Lee E.Y., Weiss H.L., Wang Q, & Evers B.M. (2021). Ketogenesis alleviates TNFα-induced apoptosis and inflammatory responses in intestinal cells. Free radical biology & medicine, 172, 90-100.
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4

Quantification of PPARγ Transcriptional Activity

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HCASMC were stimulated with E2 (10 nM) or DMSO (0.1% (v/v)) for 24 h, and the nuclear fraction was isolated using the nuclear extraction kit by Abcam (Cambridge, United Kingdom). Cells were lysed in hypotonic buffer, and nuclear protein was extracted using nuclear extraction buffers, provided with the kit. Transcription factor binding activity to dsDNA was studied using the PPARγ transcription factor assay kit (Abcam). The assay is based on the enzyme-linked immunosorbent assay (ELISA) principle. Nuclear protein fractions were pipetted onto a 96-well plate that had been coated with dsDNA containing the recognition sequence for PPARγ, PPAR response element (PPRE). Adherent PPARγ was visualised by the appropriate primary and HRP-conjugated secondary antibodies, the latter of which catalysed a colorimetric reaction. Light extinction at 450 nm was quantified using a microplate reader (Infinite M200, Tecan, Männedorf, Switzerland).
Jehle J., Tiyerili V., Adler S., Groll K., Nickenig G, & Becher U.M. (2020). Atheroprotective effects of 17β-oestradiol are mediated by peroxisome proliferator-activated receptor γ in human coronary artery smooth muscle cells. Archives of Medical Sciences. Atherosclerotic Diseases, 5, e118-e126.
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5

Adipokine and PPARγ Profiling in Cells

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To perform, the experiment cells were seeded into a 6-well plate at a density of 2 × 105 cells/well. On the last day of cells treatment the medium was collected and protein concentrations of adiponectin (Adiponectin Mouse ELISA Kit, Abcam, Cambridge, GB), leptin (Leptin Mouse ELISA Kit, Abcam, Cambridge, GB), Il6 (Mouse IL6 ELISA kit, Biorbyt Ltd., Cambridge, GB) and TNFα (Mouse TNF alpha ELISA kit, Biorbyt Ltd., Cambridge, GB), were determined using ELISA kits, following the manufacturer’s instructions. The PPARγ protein level present in the nuclear fraction of the cellular lysates was determined with the PPARγ Transcription Factor Assay Kit (Abcam, Cambridge, GB).
Zakłos-Szyda M., Pietrzyk N., Szustak M, & Podsędek A. (2020). Viburnum opulus L. Juice Phenolics Inhibit Mouse 3T3-L1 Cells Adipogenesis and Pancreatic Lipase Activity. Nutrients, 12(7), 2003.
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6

PPARγ Transcriptional Activity Assay

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Huh7 and HepG2 cells were seeded into 100 mm dishes and allowed to grow to confluency of 80%. Following treatment, nuclear and cytosolic fractions were isolated using an extraction kit (Abcam) according to the manufacturer's instructions. For experiments designed to examine the effect of GDF10 on PPARγ transcriptional activity, HepG2 cells were pre-treated with rhGDF10 for 24 h, and subsequently treated with CGTZ for 1 h prior to cellular fractionation. PPARγ transcription factor assay kit (Abcam) was carried out according to manufacturer's instructions. Briefly, 120 μg of protein isolated from nuclear extracts was added to each well of a 96-well plate coated with a double-stranded DNA sequence containing peroxisome proliferator response element (PPRE). Following an over-night incubation, anti-PPARγ primary antibody was added, followed by an HRP-conjugated secondary antibody. PPARγ binding to the PPRE was detected using a spectrophotometer at a wavelength of 450 nm.
Platko K., Lebeau P.F., Byun J.H., Poon S.V., Day E.A., MacDonald M.E., Holzapfel N., Mejia-Benitez A., Maclean K.N., Krepinsky J.C, & Austin R.C. (2019). GDF10 blocks hepatic PPARγ activation to protect against diet-induced liver injury. Molecular Metabolism, 27, 62-74.
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7

Quantifying Intracellular PPARγ Activity

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PPARγ Transcription Factor Assay Kit (Abcam, ab133101) was used to detect intracellular PPARγ-DNA binding activity. Firstly, nuclear extracts of the cells were prepared using the Nuclear Extraction Kit (Abcam, ab113474), and the resultant nuclear proteins were added to wells precoated with a specific double-stranded DNA sequence containing the peroxisome proliferator response element. Following that, specific primary antibodies and horseradish peroxidase-conjugated secondary antibodies were added according to the instructions. Finally, after adding the developing and halting solutions, the absorbance at 450 nm was determined using a microplate reader.
Ning Z., Guo X., Liu X., Lu C., Wang A., Wang X., Wang W., Chen H., Qin W., Liu X., Zhou L., Ma C., Du J., Lin Z., Luo H., Otkur W., Qi H., Chen D., Xia T., Liu J., Tan G., Xu G, & Piao H.L. (2022). USP22 regulates lipidome accumulation by stabilizing PPARγ in hepatocellular carcinoma. Nature Communications, 13, 2187.
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8

PPAR-γ Transcriptional Activity Assay

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The transcriptional activity of PPAR-γ was assessed using the purchased PPAR-γ transcription factor assay kit (Abcam, Cambridge, USA). After preparation of the nuclear extract from the left kidneys, according to the method described by the manufacturer and using the nuclear extraction kit (Abcam, Cambridge, USA), the nuclear extract was added to the provided wells coated with specific oligonucleotide sequences. A primary polyclonal anti-PPAR-γ antibody was then added, followed by the addition of horseradish peroxidase-conjugated antibody and the 3,3′,5,5′ -tetramethylbenzidine substrate. The absorbance of the developed color was read at 450 nm using a microplate reader.
Ragab D., Abdallah D.M, & El-Abhar H.S. (2014). Cilostazol Renoprotective Effect: Modulation of PPAR-γ, NGAL, KIM-1 and IL-18 Underlies Its Novel Effect in a Model of Ischemia-Reperfusion. PLoS ONE, 9(5), e95313.
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9

PPARγ DNA-binding Affinity Assay

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Nuclear protein extraction was performed using a Nuclear Extraction Kit (Abcam), following the manufacturer’s instructions. Nuclear extracts were collected on each day of adipogenesis. Protein concentration was measured with a Qubit Protein Assay Kit (Thermo Fisher Scientific) on a Qubit 2.0 Fluorometer (Invitrogen). PPARγ DNA-binding affinity was assessed using a PPARγ transcription factor assay kit (Abcam). The experiment was performed twice—initially in duplicate and then in triplicate. The protein concentrations were equalized and 10 μl of nuclear extract was added to the wells, coated with a specific DNA sequence containing the peroxisome proliferator response element, and incubated overnight at 4 °C. Washing, incubation with antibodies (primary polyclonal anti-PPARγ antibody and goat anti-rabbit HRP conjugate secondary antibody), and color development procedures were performed in line with the manufacturer’s protocol. The absorbance was read at 450 nm using a Synergy2 device (Biotek).
Stachecka J., Nowacka-Woszuk J., Kolodziejski P.A, & Szczerbal I. (2019). The importance of the nuclear positioning of the PPARG gene for its expression during porcine in vitro adipogenesis. Chromosome Research, 27(3), 271-284.
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10

PPAR-γ Activity Quantification in HAECs

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Cultured HAECs were lysed in a lysis buffer (pH, 7.4) 10 μmol/l Tris-HCl, 0.5 mmol/l NaCl in 1μmol/l ethylenediaminetetraacetic acid (EDTA), 0.05% SDS, 0.5% Triton X-100, supplemented with 1 μmol/l phenylmethanesulfonyl fluoride (PMSF). The pellets were collected after centrifugation at 15,000 g for 10 minutes at 4°C. A PPAR-γ transcription factor assay kit (Abcam, Cambridge, MA, USA) was used to determine the PPAR-γ activity by measuring the absorbance at 450 nm.
Shou X., Zhou R., Zhu L., Ren A., Wang L., Wang Y., Zhou J., Liu X, & Wang B. (2018). Emodin, A Chinese Herbal Medicine, Inhibits Reoxygenation-Induced Injury in Cultured Human Aortic Endothelial Cells by Regulating the Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) and Endothelial Nitric Oxide Synthase (eNOS) Signaling Pathway. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 24, 643-651.
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