The assay measures PPARγ binding activity in nuclear extract. It was measured in aortic tissue. Aortic nuclear fraction was obtained using NE-PER extraction reagents kit (Thermo Scientific, Rockford, IL, USA). Later, nuclear protein obtained was incubated in ELISA plate, containing PPAR response element (PPRE)-DNA sequence, following the instructions from manufacturer (PPARγ transcription factor assay kit, Cayman Chemicals, Ann Arbor, MI, USA). The colored product was spectrophotometrically measured at 450 nm. The PPARγ activity is expressed as absorbance/mg of protein quantified.
Ne per extraction reagents kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NE-PER extraction reagents kit is a product manufactured by Thermo Fisher Scientific. It is designed to facilitate the extraction and separation of nuclear and cytoplasmic proteins from cell or tissue samples. The kit provides the necessary reagents and buffers to perform this extraction process effectively.
Automatically generated - may contain errors
Lab products found in correlation
Pparγ transcription factor assay kit, by Cayman Chemical (1 mentions)
Atm pser 1981, by Cell Signaling Technology (1 mentions)
Ecl substrate, by Merck Group (1 mentions)
P53 pser15, by Cell Signaling Technology (1 mentions)
Tris hcl gel, by Bio-Rad (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
5 protocols using ne per extraction reagents kit
1
PPAR-γ Activity in Aortic Tissue
2
Comprehensive Multi-Omics Analysis Protocol
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RNA isolation, quantitative reverse transcription PCR (qRT‐PCR), Western blot (WB) and immunohistochemistry (IHC) were performed with the standard method and previously described. 24 , 25 For membrane, nuclear and cytoplasmic extraction, cells were subjected to Mem‐PER and NE‐PER Extraction Reagents Kit (89 842 and 78 833, Thermo) according to the manufacturer's instruction. The PCR primers and antibodies are shown in Tables S2 and S3 .
3
Extraction of Nuclear and Cytoplasmic Proteins
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Nuclear and cytoplasmic protein fraction contained in 40 mg of ischemic left ventricle were extracted using NE-PER extraction reagents kit (Thermo Scientific, Rockford, IL, USA) following previous reports [21 (link)] and manufacturer’s instructions.
4
Quantifying Viral Particle Localization
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NB-324K cells were infected as described for virus uptake. At various times post-infection, cells were harvested and a nucleocytoplasmic fractionation was performed using an NE-PER extraction reagents kit (ThermoScientific, Waltham, MA, USA) according to the manufacturer’s instructions. The number of full viral particles was determined in each fraction by real-time qPCR.
5
Western Blot Analysis of DNA Damage Response
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Protein content was measured with BCA assay (Pierce), 5–30 µg was subjected to SDS/PAGE on 4–20% acrylamide gel (Thermo Scientific) followed by transfer to nitrocellulose membrane. When detecting ATM, 7.5% Tris-HCl gel (Bio-Rad) was used, and protein was transferred to PVDF membrane (Millipore) overnight at 30 V. Membranes were blocked with 5% milk in TBS-T (Tween 0.1% wt/vol), and probed with the following antibodies: p53 pSer15 (1∶1000, Cell Signaling), p53 (1∶1000, Cell Signaling), ATM pSer1981 (1∶1000, Cell Signaling), α-tubulin (1∶2000, Santa Cruz), ERK2 (1∶2000, Santa Cruz), dCK (Clone 9D4, 1∶1000, Millipore). Polyclonal rabbit antibody against dCK pSer74 was a gift from Dr. Francoise Bontemps (Universite Catholique de Louvain, Brussels, Belgium). ECL substrate (Millipore) was used for detection and development on GE/Amersham film. For separating nuclear and cytoplasmic lysates, NE-PER Extraction Reagents kit was used (Thermo Scientific).
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