Samples were homogenized in a RIPA buffer (Servicebio, Biological Technology, Ltd.，Wuhan, China) containing phenylmethylsulfonyl fluoride for the total protein extraction assay according to the instructions provided by the manufacturer. An equal amount of protein (25 μg) was loaded in each lane and separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. After transfer to polyvinylidene difluoride membranes (Merck Millipore, Billerica, MA, USA), the membranes were blocked with 5% non-fat milk (Bio-Rad,Laboratories Inc., Hercules, CA, USA) at room temperature for 2 h and incubated overnight at 4°C with primary antibodies. The following primary antibodies were purchased from Proteintech (Wuhan, China):β-actin (60008–1-Ig), β-catenin (17565–1-AP), AKT (10176–2-AP), p-AKT(S473) (66444–1-Ig),E-cadherin (20874–1-AP), Vimentin (10366–1-AP), P21 (10355–1-AP), and CCNB2 (21644–1-AP) Of note, p-GSK3β (Ser9) (D85E12) was purchased from CST (USA). The membranes were washed and incubated in horseradish peroxidase-conjugated goat anti-mouse or goat anti-mouse or anti-rabbit immunoglobulin G secondary antibody. Protein bands were visualized using an ChemiDocTM XRS + with image LabTM Software (BIORAD, USA).
Chemidoc xrs with image labtm software
Manufactured by Bio-Rad
Sourced in United States
The ChemiDoc XRS+ with Image Lab Software is a high-performance imaging system designed for capturing and analyzing images of electrophoresis gels, Western blots, and other samples. The system features a sensitive CCD camera, a motorized sample stage, and a range of filter and illumination options to optimize image quality. The included Image Lab software provides a comprehensive suite of tools for image acquisition, processing, and analysis. The system is suitable for a variety of applications in molecular biology, biochemistry, and other life science research areas.
Automatically generated - may contain errors
Lab products found in correlation
Non fat milk, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Hybridization nitrocellulose filter membrane, by Merck Group (1 mentions)
Anti col1, by Abcam (1 mentions)
Protease inhibitor cocktail, by Selleck Chemicals (1 mentions)
Anti znf451, by Proteintech (1 mentions)
Bca protein assay reagent kit, by Applygen (1 mentions)
Anti α sma, by Boster Bio (1 mentions)
Anti gapdh, by ZSGB-BIO (1 mentions)
Phosphatase inhibitor, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Primary antibody diluent, by Beyotime (1 mentions)
4 protocols using chemidoc xrs with image labtm software
1
Western Blot Analysis of Protein Expression
2
Quantifying SLR1 Protein Abundance
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For measuring SLR1 abundance, total proteins were extracted using a protein extraction buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2% SDS, 1 mM EDTA, 1 mM dithiothreitol, and 1 μM PMSF), and then separated in a 10% SDS-PAGE gel. After the protein sample was transferred from the SDS-PAGE gel to a HATF (Hybridization Nitrocellulose Filter) membrane (Merck Millipore, Ireland), immunodetection of SLR1 was performed with a rabbit anti-SLR1 primary antibody and an anti-IgG-HRP secondary antibody (Abmart, China). The anti-SLR1 primary antibody was kindly provided by Donglei Yang17 (link). HRP signal was detected using the SuperSignalTM West Pice PLUS kit (Thermo Scientific, USA). Images were captured using ChemiDocTM XRS + with Image LabTM software (BIO-RAD).
3
Western Blot Analysis of Lung Proteins
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Mouse lung tissues or cells were lysed with RIPA Lysis Extraction Buffer (Beyotime Technology) along with protease inhibitor cocktail (Selleck). The total protein concentration was determined by a BCA protein assay reagent kit (Applygen Technologies Inc.) according to the manufacturer’s protocol. Total protein (20 µg) was loaded and separated by 10% SDS‒PAGE and then transferred to a PVDF membrane. Membranes were blocked with 5% skim milk in TBST buffer for 1 h and then incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The following antibodies were used: anti-ZNF451 (Proteintech), anti-α-SMA (BOSTER), anti-Col1 (Abcam), and anti-GAPDH (ZSGB BIO). The signaling was visualized using a ChemiDocTM XRS + with Image LabTM Software (Bio-Rad, Hercules, California, USA) with an ECL kit (Tanon).
4
Protein Expression Analysis in Liver Tissue
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Sequence
Western blot
Protein were lysed from the the liver tissues and HSCs using radioimmuno-precipitation assay buffer combination with a protease inhibitor PMSF (Sigma, USA) and phosphatase inhibitors (Beyotime, China). Protein concentrations were measured by the BCA quantitative kit. Then protein was separated by SDS-PAGE, electro-transferred to nitrocellulose lter membrance, blocked with Quickblock™ Western block kit (Genscript, USA), and probed with the following antibodies overnight at 4℃. (diluted with primary antibody diluent, Beyotime, China). The membrane was subsequently incubated with HRP-conjugated Rabbit or Mouse secondary antibody (1:7500, Abcam) for 1h at room temperature. Speci c signals were detected using the enhanced ECL kit (Thermo Scienti c, USA). The chemiDocTM XRS + with Image LabTM Software (BIO-RAD, USA) was used for development.
Western blot
Protein were lysed from the the liver tissues and HSCs using radioimmuno-precipitation assay buffer combination with a protease inhibitor PMSF (Sigma, USA) and phosphatase inhibitors (Beyotime, China). Protein concentrations were measured by the BCA quantitative kit. Then protein was separated by SDS-PAGE, electro-transferred to nitrocellulose lter membrance, blocked with Quickblock™ Western block kit (Genscript, USA), and probed with the following antibodies overnight at 4℃. (diluted with primary antibody diluent, Beyotime, China). The membrane was subsequently incubated with HRP-conjugated Rabbit or Mouse secondary antibody (1:7500, Abcam) for 1h at room temperature. Speci c signals were detected using the enhanced ECL kit (Thermo Scienti c, USA). The chemiDocTM XRS + with Image LabTM Software (BIO-RAD, USA) was used for development.
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