Proteins were separated on a sodium dodecyl sulfate-polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Bio-Rad, USA). After blocking with 5% bovine serum albumin for 50 min at room temperature, the membranes were incubated with primary antibodies against ZRANB2 (1 : 2000, ab82132, Abcam, Shanghai, China) and the loading control GAPDH (1 : 500, ab8245, Abcam) at 4°C overnight. The membranes were incubated with the horseradish peroxidase-labeled secondary antibodies for 1 h at room temperature in dark. Signal was detected using an enhanced chemiluminescence western blotting substrate (Bio-Rad, USA). The band density was quantified using the ImageJ software.
Enhanced chemiluminescence western blotting substrate
Manufactured by Bio-Rad
Sourced in United States
Enhanced chemiluminescence western blotting substrate is a reagent used in the Western blotting technique to detect and quantify specific proteins in a sample. It generates a luminescent signal when it reacts with the enzyme-labeled antibodies bound to the target proteins, allowing their visualization and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene uoride membrane, by Bio-Rad (3 mentions)
Ab8245, by Abcam (2 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (2 mentions)
Ab174963, by Abcam (1 mentions)
Ab234099, by Abcam (1 mentions)
Bicinchoninic acid kit, by Thermo Fisher Scientific (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab32572, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (1 mentions)
Anti bcl2 associated x bax, by Santa Cruz Biotechnology (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary abs, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
P c jun n terminal kinase jnk, by Cell Signaling Technology (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
8 protocols using enhanced chemiluminescence western blotting substrate
1
Western Blot Analysis of ZRANB2 Protein
2
Western Blot Protein Detection Protocol
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Proteins were separated on a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (Bio-Rad, USA). The membranes were blocked with 5% bovine serum albumin (BSA) for 50 min at room temperature before incubation with primary antibody at 4 °C overnight. The membranes were incubated with secondary antibody conjugated to horseradish peroxidase, followed by signal detection using an enhanced chemiluminescence western blotting substrate (Bio-Rad, USA). The primary antibodies used here are listed in Supplementary Table 2 .
3
Wnt Signaling Pathway Protein Analysis
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Total proteins from H9C2 cells lysed using the radioimmunoprecipitation assay buffer were quantified using a bicinchoninic acid kit (Thermo Scientific, USA). Proteins (20 µL) were loaded and subjected to gel electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Membranes were then exposed to the blocking buffer for 1 h at room temperature and incubated with Wnt3a (ab234099, 1:500; Abcam, UK), Wnt5a (ab174963, 1:500; Abcam), β-catenin (ab32572, 1:500; Abcam), and GAPDH (ab8245, 1:1000; Abcam) antibodies with agitation at 4°C overnight. Subsequently, the membranes were incubated with secondary antibodies (ab6721, 1:2000; Abcam) for 1 h. Enhanced chemiluminescence western blotting substrate (Bio-Rad, USA) was used to detect the signals [25 (link)].
4
Western Blot Analysis of Mitochondrial Proteins
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Proteins were extracted from tissues and cells using radioimmunoprecipitation assay buffer (catalog No. 8900; Thermo Fisher Scientific) containing a cocktail of protease and phosphatase inhibitors (GenDEPOT, Baker, TX, USA). The protein lysates (20 μg) were separated using 10%–12% SDS-PAGE and transferred (100 volts, 90 min) onto polyvinylidene difluoride membranes (GE Healthcare, Solingen, Germany). Membranes were blocked using 5% skim milk with Tris-buffered saline containing 0.1% Tween 20 for 1 h and then probed with the primary Abs, anti-Miro-1 (catalog No. NBP1-89011; Novus Biologicals, Centennial, CO, USA), anti-Bcl-2 (catalog No. sc-7382; Santa Cruz Biotechnology), and anti-Bcl-2-associated X (Bax) (catalog No. sc-7480; Santa Cruz Biotechnology), overnight at 4°C. The membranes were incubated with horseradish peroxidase-conjugated secondary Abs (anti-rabbit, catalog No. 1706515 or anti-mouse, catalog No. 1706516; Bio-Rad, Hercules, CA, USA) for 1 h at 20–25°C. The signal was developed using an enhanced chemiluminescence western blotting substrate (Bio-Rad). Quantification was performed using the ImageJ software and normalized against β-actin.
5
Western Blot Analysis of Signaling Proteins
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Cells were lysed in radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The lysates were collected by centrifugation at 13,000× g for 30 min at 4 °C. Protein quantification was performed using the bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Equal quantities of total protein were separated using Mini-PROTEAN TGX™ Precast Protein Gels and transferred onto polyvinylidene fluoride membranes using Trans-Blot Turbo Transfer System (Bio-rad, Hercules, CA, USA). The membranes were blocked with 5% nonfat milk and incubated with primary antibodies against c-Fos, NFATc1 (Santa Cruz Biotechnology, Dallas, TX, USA), p-extracellular signal-regulated kinase (ERK), ERK, p-c-Jun N-terminal kinase (JNK), JNK, p-p38, p38, p-IκBα, IκBα, and β-actin (Cell Signaling Technology, Danvers, MA, USA). After incubation with horseradish peroxidase-conjugated secondary antibodies, specific bands were detected with a LAS-4000 image analyzer (Fujifilm, Tokyo, Japan) and Image Gauge software V3.0 (Fujifilm, Tokyo, Japan) using an enhanced chemiluminescence western blotting substrate (Bio-Rad, Hercules, CA, USA).
6
Quantitative Western Blot Protein Analysis
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Proteins were separated on a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto polyvinylidene uoride membranes (Bio-Rad, USA). The membranes were blocked with 5% bovine serum albumin (BSA) for 50 min at room temperature before incubation with primary antibody at 4 °C overnight. The membranes were incubated with secondary antibody conjugated to horseradish peroxidase, followed by signal detection using an enhanced chemiluminescence western blotting substrate (Bio-Rad, USA). The primary antibodies used here are listed in Supplementary Table 2.
7
Western Blot Protein Analysis
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Protein were separated on an SDS-polyacrylamide gel followed by transferring onto polyvinylidene uoride membranes (Bio-Rad, USA). The membranes were blocked with 5% BSA for 50 minutes at room temperature before incubated with primary antibody in 4 ℃ overnight. The membranes were incubated with secondary antibody conjugated to horseradish peroxidase, followed by signal detection by enhanced chemiluminescence Western blotting substrate (Bio-Rad, USA). The primary antibodies used here were listed in supplementary Table 2.
8
Western Blot Protein Analysis
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Protein were separated on an SDS-polyacrylamide gel followed by transferring onto polyvinylidene uoride membranes (Bio-Rad, USA). The membranes were blocked with 5% BSA for 50 minutes at room temperature before incubated with primary antibody in 4 ℃ overnight. The membranes were incubated with secondary antibody conjugated to horseradish peroxidase, followed by signal detection by enhanced chemiluminescence Western blotting substrate (Bio-Rad, USA). The primary antibodies used here were listed in Supplementary Table 2.
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