Kidney tissues were homogenized in M-PER tissue protein extraction reagent (Thermo Scientific, Waltham, MA, USA) supplemented with protease inhibitor mixture (Calbiochem, Darmstadt, Germany) at 4°C for 30 min, and then centrifuged at 4000 g for 15 min, the supernatants were collected. The protein concentrations were measured using BCA assay (Thermo Scientific, Waltham, MA, USA) and equalized to 4 μg/μl using extraction reagent. The TNF-α, TGF-β, IL-1β and IL-6 levels of the kidney were determined by commercial enzyme linked immunosorbent assay (ELISA) kits (Bender MedSystems GmbH, Vienna, Austria) according to the manufacturer's instructions.
M per tissue protein extraction reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
M-PER tissue protein extraction reagent is a commercially available solution designed for the efficient extraction of proteins from various tissue samples. It is a gentle, non-denaturing reagent that preserves the native structure and function of the extracted proteins.
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Lab products found in correlation
4 protocols using m per tissue protein extraction reagent
1
Kidney Protein Extraction and Cytokine Analysis
2
Western Blot Analysis of ERα and ERβ
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Treated cells were washed with PBS, trypsinized, and centrifuged at 5000g. The pellets were washed with PBS and homogenized in M-PER tissue protein extraction reagent (Thermo Scientific; Waltham, MA) supplemented with protease and phosphatase inhibitors. Denatured proteins (5 µg) were separated in 10% SDS-PAGE gels, transferred to a PVDF membrane (Millipore; Billerica, MA) and incubated with antibodies to ERα (1:1000, Santa Cruz; Dallas,TX) and ERβ (1:1000, Millipore, Billerica, MA). β-actin was used as a gel loading control. The blots were developed with enhanced chemiluminescence (ECL Clarity kit, Bio-Rad). Bands were visualized on a polyvinylidene difluoride membrane and analyzed by LabWorks 4.5 software on a UVP Bioimaging System. Quantification of results was performed by densitometry and the results analyzed as total integrated densitometric values (arbitrary units).
3
Western Blot Analysis of p53 and MDM2 in MCF7 Cells
4
Western Blot Analysis of Inflammatory Markers
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Protein samples were isolated from tissue using M-PER tissue protein extraction reagent (Thermo Fisher Scientific), EDTA, and protease inhibitor cocktail. Protein concentration was assessed using Bradford’s method and an equal concentration of protein were loaded onto gels and separated using SDS-PAGE. Proteins were transferred onto a PVDF membrane, which was blocked using 5% skim milk in TBS-0.1% Tween buffer. Membranes were incubated in the presence of primary antibodies (anti-NLRP3, anti-cleaved caspase-1, IL-1B, IL-18, TSHR, IGF-1R, or β-actin) overnight at 4°C. The membrane was then incubated with HRP-conjugated secondary antibody for 1 h at room temperature. The blots were washed thoroughly before incubation with chemiluminescence detection fluid which was further imaged using a chemiluminescent reader. Protein bands were quantified by densitometric analysis using Image J software.
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