Total protein was isolated from kidney cortex tissues using Radio Immunoprecipitation Assay Lysis buffer with protease phosphatase inhibitors and protease inhibitors obtained from Beyotime, and the extract was centrifuged at 10,000 ×g for 15 min to remove cell debris. Protein concentration was quantified using a BCA protein assay kit (Beyotime). Then, protein samples (20 μg/group) were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride membrane. After blocking with 5% skimmed milk, the membrane was incubated overnight with primary antibodies against Bax (ab182733, 1:2000; Abcam), Nrf2 (#20733, 1:1000; Cell Signaling Technology), cleaved caspase-3 (#9661, 1:1000; Cell Signaling Technology), Sirt1 (ab189494, 1:1000; Abcam), Bcl-2 (ab196495, 1:2000; Abcam), β-actin (ab8227, 1:2000; Abcam), and HO-1 (ab189491, 1:2000; Abcam) at 4 °C. Then the membranes were incubated with secondary antibodies for 2 h at room temperature. The bands were visualized using an enhanced chemiluminescent reagent (Yeasen, Shanghai, China). The immunoblot images were evaluated by ImageJ software.
Enhanced chemiluminescent reagent
Manufactured by Yeasen
Sourced in China
The Enhanced chemiluminescent reagent is a laboratory product designed for use in various biochemical and molecular biology applications. It serves as a reagent for the detection and quantification of proteins or other biomolecules through chemiluminescent reactions.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Ab182733, by Abcam (1 mentions)
Ab189494, by Abcam (1 mentions)
Ab196495, by Abcam (1 mentions)
Ab189491, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
2 protocols using enhanced chemiluminescent reagent
1
Quantitative Western Blot Analysis of Kidney Proteins
2
GPT Protein Expression Analysis in GC Cells
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The protein level of GPT was measured using Western blotting as described previously[17 (link)]. The total protein was isolated from GC cells (AGS, MKN45, MKN28 and HGC27) and the normal immortalized cells (GES1) using RIPA lysis buffer containing protease phosphatase cocktail obtained from Beyotime (Shanghai, China). The extract was centrifuged at 10000 × g for 15 min to remove the cell debris. The protein concentration was quantified using a bicinchoninic acid (BCA) protein assay kit (Beyotime). Thereafter, the protein samples (20 μg per lane) were resolved using 10% Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% skimmed milk for 1 h, the membrane was incubated overnight with the primary antibodies against GPT (PAS-29600, 1:1000; Thermo Fisher Scientific, Shanghai, China) and GAPDH (ab8245, 1:2000; Abcam) at 4 ℃. Subsequently the membranes were incubated with secondary antibodies for 2 h at the room temperature. The bands were visualized using an enhanced chemiluminescent reagent (Yeasen, Shanghai, China). The immunoblot images were quantified by ImageJ software.
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