Total protein from renal tissues was extracted with ice-cold RIPA lysis buffer (Beyotime, Shanghai, China). The lysate was centrifuged at 10,000 × g for 15 min at 4 °C. The protein concentrations were determined with a BCA Protein Assay Kit (Solarbio, Beijing, China). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Burlington, MA, USA). Membranes were blocked in 5% nonfat milk at room temperature for 1 h and were then incubated with primary antibodies against IL-1β, IL-6, nephrin, p-Akt (all at a 1:1000 dilution), IV-C, MCP-1, p-p38 MAPK, TNF-ɑ (all at a 1:500 dilution), α-SMA (1:100 dilution) and GAPDH (1:5000 dilution) respectively overnight at 4 °C and were then washed. After incubation with the appropriate secondary antibodies (1:1000 dilution) for 1 h at room temperature, target protein bands were detected with enhanced chemiluminescence substrate (Solarbio, Beijing, China). Each WB analysis was performed three times. Semiquantitative analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Enhanced chemiluminescence substrate
Manufactured by Solarbio
Sourced in China
The Enhanced chemiluminescence substrate is a laboratory reagent used to detect and quantify protein targets in Western blot analyses. It provides a sensitive and stable chemiluminescent signal that can be detected using specialized imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Ab68153, by Abcam (1 mentions)
Ab52903, by Abcam (1 mentions)
Ab9483, by Abcam (1 mentions)
Ab179800, by Abcam (1 mentions)
Ab216462, by Abcam (1 mentions)
Ab231303, by Abcam (1 mentions)
Ab73246, by Abcam (1 mentions)
Ab84177, by Abcam (1 mentions)
Ab32499, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Sab4503292, by Merck Group (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ab76315, by Abcam (1 mentions)
Goat anti mouse igg, by Solarbio (1 mentions)
Bca kit, by Solarbio (1 mentions)
Radioimmunoprecipitation assay buffer, by Solarbio (1 mentions)
Goat anti rabbit igg, by Solarbio (1 mentions)
3 protocols using enhanced chemiluminescence substrate
1
Renal Protein Expression Analysis
2
Western Blot Analysis of Protein Markers
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Western blot assay was performed as previously described [16 (link)]. Briefly, proteins isolated from the kidneys of mice were quantified using a BCA kit (Beyotime, Shanghai, China). The proteins were then separated by SDS/PAGE and transferred onto poly(vinylidene difluoride) membranes (Millipore, Billerica, MA, USA), which were subsequently incubated overnight at 4 °C with the primary antibodies (dilution 1 : 1000). Antibodies against NGAL (ab216462), PRMT1 (ab73246), GAPDH (ab9483), Smad3 (ab84177), p‐Smad3 (ab52903), E‐cadherin (E‐cad) (ab231303), pro caspase‐3 (ab32499), COX‐2 (ab179800), p‐STAT3 (ab76315), and STAT3 (ab68153) were purchased from Abcam (Cambridge, UK). Antibody against sIL‐6R (23457) was purchased from Proteintech (Rosemont, IL, USA). Antibody against cleaved caspase‐3 (SAB4503292) was purchased from Sigma‐Aldrich. The membranes were then incubated with the secondary antibody coupled with horseradish peroxidase at room temperature for 30 min. The bands of proteins were visualized via enhanced chemiluminescence substrate (Solarbio, Beijing, China). GAPDH served as an internal control.
3
Protein Expression Analysis in EC Cells
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Total proteins were obtained from EC cells through radio-immunoprecipitation assay buffer (R0010; Solarbio). Next, the protein concentration was quantitated by the BCA kit (PC0020; Solarbio). Then, the proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto membranes (2215; Millipore, USA). After being blocked with 5% non-fat milk, membranes were reacted with primary antibodies against PCNA (ab92552, 29 kDa, ½,000), E-cadherin (ab231303, 97 kDa, 1 µg/mL), N-cadherin (ab18203, 100 kDa, 1 µg/mL), Vimentin (ab20346, 54 kDa, 1/1,000), VEGFR-1 (ab32152, 151 kDa, 1/2,000), VEGFR2 (ab11939, 151 kDa, 2 µg/mL), epidermal growth factor receptor (EGFR) (ab52894, 175 kDa, 1/2,000), and GAPDH (ab181602, 36 kDa, 1/10,000). Subsequently, the membranes were incubated with the appropriate secondary antibodies goat anti-rabbit IgG (ab205718, 1/5,000) as well as goat anti-mouse IgG (ab205719, 1/5,000), followed by being exposed to the Enhanced Chemiluminescence Substrate (PE0010; Solarbio) for visualization. All the antibodies were bought from Abcam (USA).
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