The liver tissue and cell lysates were homogenized in mammalian protein extraction buffer (Sigma Chemical Co., St. Louis, MO. USA) in the presence of protease inhibitor cocktail (Sigma) and phenyl methane sulfonyl fluoride (Sigma, PMSF). The lysates were centrifuged at 12,000 rpm at 4 °C for 20 min. The protein contents of the supernatants were determined using protein assay dye reagent concentrate (Bio-Rad Laboratories, Hercules, CA, USA) based on the manufacturer’s instructions. The same concentration of protein was separated electrophoretically by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose blotting membranes (Amersharm, GE Healthcare Life Science, Germany). The antibodies used for western blotting included anti-SREBP-1 (1:1000; Abcam, Cambridge, UK), anti-ACC (1:1000; Cell Signaling Technology), anti-FAS (1:1000; Cell Signaling Technology), anti-AMPKα (1:1000; Cell Signaling Technology), anti-phospho-AMPKα (Thr172) (1:1000; Cell Signaling Technology), and anti-β-actin (1:2500; Abcam). The protein bands were visualized following an enhanced chemiluminescence method using an ELC kit (Millipore, USA). The bands were quantified using Quantity 1 version 4.6.7 software (Bio-Rad Laboratories).
Mammalian protein extraction buffer
Manufactured by Merck Group
Sourced in United States
Mammalian protein extraction buffer is a solution designed to facilitate the extraction and isolation of proteins from mammalian cell samples. It is a balanced formulation that helps maintain the structural integrity and solubility of proteins during the extraction process.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay dye reagent concentrate, by Bio-Rad (3 mentions)
Amersharm, by GE Healthcare (2 mentions)
Quantity 1 version 4, by Bio-Rad (2 mentions)
Elc kit, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti acc, by Cell Signaling Technology (1 mentions)
Anti ampkα, by Cell Signaling Technology (1 mentions)
Anti fas, by Cell Signaling Technology (1 mentions)
Anti srebp1, by Abcam (1 mentions)
Anti phospho ampkα thr172, by Cell Signaling Technology (1 mentions)
β actin 13e5 rabbit mab, by Cell Signaling Technology (1 mentions)
Parp 46d11 rabbit mab, by Cell Signaling Technology (1 mentions)
Ampkα d63g4 rabbit mab, by Cell Signaling Technology (1 mentions)
Bax rabbit mab, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3 asp175 5a1e rabbit mab, by Cell Signaling Technology (1 mentions)
Caspase 3 rabbit mab, by Cell Signaling Technology (1 mentions)
Cox 2, by Cell Signaling Technology (1 mentions)
4 hydroxynonenal 4 hne, by Abcam (1 mentions)
Anti claudin 1, by Thermo Fisher Scientific (1 mentions)
3 protocols using mammalian protein extraction buffer
1
Protein Expression Analysis in Liver Tissue
2
Western Blot Analysis of Protein Biomarkers
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The cell lysates were homogenized in mammalian protein extraction buffer (Sigma). The resulting lysates were centrifuged at 12,000 rpm, and 4°C for 15 min, and the protein contents were measured using a protein-assay dye reagent concentrate (Bio-Rad Laboratories, Hercules, CA, USA). Subsequently, 10–15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed, followed by transfer onto nitrocellulose blotting membranes (Amersham, GE Healthcare Life Science, Germany). The antibodies used for immunoblotting are as follows: Bax rabbit mAb (1:1,000; Cell Signaling Technology), Bcl2 (D17C4) rabbit mAb (1:1,000; Cell Signaling Technology), inducible nitric oxide synthase (iNOS; NOS2) (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), AMPKα (D63G4) rabbit mAb (1:1,000; Cell Signaling Technology), Phospho-AMPKα (Thr172) (D4D6D) rabbit mAb (1:1,000; Cell Signaling Technology), caspase-3 rabbit mAb (1:1,000; Cell Signaling Technology), cleaved caspase-3 (Asp175) (5A1E) rabbit mAb (1:1,000; Cell Signaling Technology), PARP (46D11) rabbit mAb (1:1,000; Cell Signaling Technology), and β-actin (13E5) rabbit mAb (1:2,500; Cell Signaling Technology). The protein bands were visualized using an enhanced chemiluminescence method with an ELC kit (Millipore, Billerica, MA, USA) and were quantified using Quantity 1 v.4.6.7 (Bio-Rad).
3
Protein Extraction and Western Blot Analysis
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Proteins were extracted with the mammalian protein extraction buffer (Sigma Chemical Co., St. Louis, MO, USA) containing the protease inhibitor cocktail (Sigma) and phenyl methane sulfonyl fluoride (PMSF, Sigma). Protein concentrations in supernatants collected after centrifugation of protein lysates were established using protein assay dye reagent concentrates (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein were split by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersharm, GE Healthcare Life science, Germany). Primary antibodies were reacted with anti-occludin (1:1000; Invitrogen, USA), anti-claudin-1 (1:1000; Invitrogen), anti-ZO-1 (1:1000; Invitrogen), iNOS (1:500; Santa cruz, USA), COX2 (1:1000; Cell signalling), 4-hydroxynonenal (4HNE) (1:1000; Abcam, UK), AMPK (1:1000; Cell signalling), phospho-AMPK (1:1000; Cell signalling), and anti-β-actin (1:2500; Abcam, Cambridge, UK) at 4 °C overnight, respectively. After secondary antibody reaction for 2 h at room temperature, target protein bands were visualized by an enhanced chemiluminescence method (ECL, Millipore, Boston, MA, USA). Quantity of luminescence was performed using Quantity 1 version 4.6.7 software (Bio-Rad Laboratories, Hercules, CA, USA).
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