Liver tissues from WT and C6orf120–/– rats were immediately excised and mixed with tissue protein lysis buffer (Beyotime, Shanghai, China) containing 1 mM phenylmethane sulfonyl fluoride (Beyotime Biotechnology, ST506). After centrifugation, the proteins were denatured and the protein was loaded onto sodium dodecyl sulfate-polyacrylamide gels (10%) for electrophoretic separation and then transferred onto Immobilon polyvinylidene fluoride membranes (Sigma; EMD Millipore, Billerica, MA, USA; cat. no. P3313). After blocking with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST), the membranes were incubated at 4°C overnight with primary antibodies against C6orf120, NLRP3, caspase 1, IL-1β, nuclear factor-κB, Bcl-2, and Bax (Abcam), as well as cleaved caspase-3, p-c-Jun N-terminal kinase (Cell Signaling Technology). The membranes were washed with TBST three times. Then, they were incubated with secondary antibodies (ZF-0316, ZSGB-BIO, Beijing, China) for 2 h at room temperature. The finally obtained immunoreactive protein bands were imaged using the Tanon Imaging system (Tanon Science and Technology Co., Ltd., Shanghai, China).
Tissue protein lysis buffer
Manufactured by Beyotime
Sourced in China
Tissue protein lysis buffer is a solution designed to efficiently extract and solubilize proteins from biological tissues. It is a key component in the protein extraction and analysis workflow, facilitating the release of proteins from complex cellular environments.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using tissue protein lysis buffer
1
Liver Protein Expression Analysis
2
Western Blot Analysis of Adiponectin Signaling
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Frozen tissues (100 mg) from each sample were homogenized for 10 min with tissue protein lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) using a homogenizer, chilled on ice for 30 min, and then centrifuged at 10,000 g for 5 min at 4°C. The protein concentration was determined using a protein assay kit (BCA, Pierce Chemical). The same amount of protein (50 µg) was separated by 8% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (Jianglai Science and Technology Co., Ltd., Shanghai, China). The membrane was blocked with 5% bovine serum albumin for 1 h at room temperature, followed by incubation with primary antibodies against APN, AdipoR1, AdipoR2, phosphorylated AMPK or total AMPK (ABCAm plc) at 4°C overnight. Then, the membrane was incubated with a corresponding horseradish peroxidase-conjugated secondary antibody (ZSGB-Bio, Beijing, China) for 2 h at room temperature. Proteins were detected using an enhanced chemiluminescence reagent (SuperSignal West Pico, Pierce Chemical). Band intensity was quantified using the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Protein expression was normalized to that of translation initiation factor 5 (Santa Cruz Biotechnology). Quantitative analysis was performed using ImageJ software (National Institutes of Health).
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