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.
Potent ecl kit
Manufactured by MultiSciences Biotech
Sourced in China
The Potent ECL kit is a laboratory equipment designed for enhanced chemiluminescence (ECL) detection. It provides a reliable and sensitive method for detecting and quantifying target proteins in Western blot analysis.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using potent ecl kit
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
Comparative Analysis of Synovium Proteins in RA and No-RA Groups
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Synovium samples were divided into the M-RA group (n = 3), F-RA group (n = 3), male No-RA group (n = 3), and female No-RA group (n = 3). Western blotting analysis was performed in the 4 groups, and the gray values were compared. The proteins were extracted from the samples, and the total protein concentration was determined using the BCA protein concentration determination kit (Multi Sciences, Hangzhou, China). The proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were sealed for 1 hour in 5% skimmed dried milk in a TBST buffer at 37°C and incubated overnight with primary antibody (Sangon Biotech, Shanghai, China) at 4°C. The membranes were then washed in TBST for 3 times and incubated with secondary antibodies at room temperature for 30 minutes. Potent ECL kit (Multi Sciences, Hangzhou, China) was used for chemiluminescence development. The ImageJ (National Institutes of Health, the United States of America) software was used to analyze the optical density of the stripe.
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