Membrane protein of liver samples was extracted with membrane protein extraction kit (Beyotime Institute of Biotechnology, China) according to the protocol. Protein concentrations were measured with a modified BCA technique. An equal amount of membrane protein (100 μg) per lane was separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the gels were transferred to polyvinylidene difluoride membranes which were blocked with Tris-buffered saline containing 5% nonfat milk. Then the membranes were incubated overnight at 4°C in Tris-buffered saline containing 0.1% Tween 20 (TBST), 5% nonfat milk, and anti-PDZK1 (at the dilution of 1 : 200). After being washed three times in TBST, the membranes were incubated with HRP-conjugated secondary antibody for 2 h at room temperature and subsequently processed for enhanced chemiluminescence (ECL) detection using potent ECL kit (Multisciences, China). Signals were detected using a chemiluminescence detection system (IS4000MM Pro, Kodak, USA). β-Actin was used as a loading control.
Is4000mm pro
Manufactured by Kodak
Sourced in United States
The IS4000MM Pro is a high-precision laboratory instrument designed for advanced imaging and spectroscopy applications. It features a powerful imaging sensor and advanced optics that deliver exceptional image quality and data accuracy. The core function of the IS4000MM Pro is to capture and analyze detailed images and spectral data for a wide range of scientific and research purposes.
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3 protocols using is4000mm pro
1
Western Blot Analysis of PDZK1
2
Hepatic Apoptosis Protein Expression
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The whole liver lysate was prepared to evaluate the expression level of Bcl-2, Bax, and cleaved caspase-3. The protein concentration was determined using the bicinchoninic acid assay and samples were stored at −80℃. Equal amounts of protein were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the gels were transferred onto polyvinylidene difluoride membranes, which were blocked with Tris-buffered saline containing 5% nonfat milk at 4℃. Next, the membranes were incubated overnight at 4℃ in solution containing 0.1% Tween 20, 5% nonfat milk, and the following primary antibodies: Bcl-2 (1:500); Bax (1:500); cleaved caspase-3 (1:1000); phosphoPI3K (1:1000); PI3K (1:1000); phosphoAkt (1:1000); Akt (1:1000); GAPDH (1:20000). After three washes in Tris-buffered saline Tween 20, the membranes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature and subsequently processed for enhanced chemiluminescence (ECL) detection using potent ECL kit (Multisciences, China) and a chemiluminescence detection system (IS4000MM Pro, Kodak, USA). GAPDH was used as an internal index.
3
Quantification of Tn-C and Fibronectin Levels
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Liver tissues were grinded and lysed in buffer (Pro-prep, iNtRON) for 30 min. After centrifugation for 5 min at 14,000 rpm, the supernatant protein was collected. The concentrations were measured by Bradford assay, and 5 ug of cell extracts were applied to each lane along with a protein marker (Santa Cruz). They were separated by standard electrophoresis using 10% acrylamide separating gels and 4% stacking gels. After that, they were transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were incubated with anti-Tn-C, Fibronectin, and actin primary antibody (Santa Cruz) at the dilution of 1:1000 and detected using a western detection solution (ECL, Amersham Life Science). The OD ratio of Tn-C and fibronectin expression was estimated by using Molecular imaging system IS-4000 MM pro (Kodak, Rochester, NY, USA).
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