After treatment, cells or kidney tissue were homogenized in RIPA lysis buffer with PMSF and protease inhibitor cocktail (Roche) for 30 min at 4°C. Total proteins were separated in an 8-10% SDS-PAGE gel and transferred onto PVDF membranes. Then, the membranes were blocked with 5% milk for 1h. After blocking with milk, the membranes were incubated with primary antibodies (PFKP, PFKM, PFKL, PKM2, ALDOB, ROCK1, RhoA, α-tubulin and GAPDH) overnight at 4°C. The next day, the membranes were incubated with a secondary antibody (Antgene, China). After washing the membranes three times, bands were revealed by an ECL chemiluminescent kit (Biosharp, China). Finally, the bands were analyzed using a ChemiDocTM MP Imaging system (Bio-Rad, USA).
Ecl chemiluminescent kit
Manufactured by Biosharp
Sourced in China
The ECL chemiluminescent kit is a laboratory reagent used for the detection and visualization of proteins through Western blotting. It contains the necessary components to generate a chemiluminescent signal, which can be captured and analyzed using imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Imagej software, by IBM (1 mentions)
Af7021, by Affinity Biologicals (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
St505, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
3 protocols using ecl chemiluminescent kit
1
Western Blot Analysis of Glycolytic Enzymes
2
Western Blot Analysis of RBMS3 Protein
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A lysis buffer containing 1 mM phenylmethylsulfonate fluoride (PMSF; No.: BL612A; Beijing Labgic Technology Co., Ltd., China) was applied to extract the total protein using RIPA (No.: P0013B; Shanghai Beyotime Biotech Co., Ltd., China). Using a fast preparation kit (No.: ZJ102; Shanghai Yazy Biomedical Technology Co., Ltd., China), after samples were electrophoretically separated on 10% SDS-PAGE gels, they were transferred to PVDF membranes for 80 min at a constant current of 200 mA. Prior to being incubated with primary antibodies against RBMS3 (1:2000; No.: DF8599; Affinity Biologicals, China) or GAPDH (1:10,000; No.: AF7021; Affinity Biologicals, China) for an entire night on a shaking table at 4 °C, for 2 h under a condition of the room temperature, 5% skim milk was applied to block the membranes. After that, they had three TBST washes (10 min each wash). The following step involved incubating an anti-rabbit secondary antibody coupled with horseradish peroxidase for 1 h under a condition of the room temperature. After that, the PVDF membranes were found using the ECL-chemiluminescent kit (No.: BL520A, Biosharp, Beijing, China) and quantified using ImageJ software from IBM Corp. (Armonk, NY, USA).
3
Western Blot Analysis of Liver Proteins
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Total proteins of liver samples were homogenized in RIPA (P0013C, Beyotime, China) lysis with Phenylmethylsulfonyl fluoride (PMSF, ST505, Beyotime, China) and phosphatase inhibitors. Protein concentrations were detected by BCA protein assay kit (P0012S, Beyotime, China). 30 μg protein samples were subjected to 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Then, the protein was transferred onto polyvinylidene fluoride (PVDF) membrane (IPVH00010, Millipore, United States). The membranes were blocking with 5% milk for 1 h. Subsequently, membranes were incubated overnight at 4°C with the specific primary antibodies (1:1000 dilutions) respectively. After washing three times, membranes were incubated with HRP-conjugated secondary antibody (1:100 dilutions) for 1 h. After washing, membranes were visualized with ECL chemiluminescent kit (BL523A, Biosharp, China). The blot values were calculated using ChemiDocTM MP Imaging system (Bio-Rad, United States).
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