Protein was extracted with Proprep (Intron Co., Seoul, South Korea) following the manufacturer’s protocol. Cytosolic protein (30 μg) was subjected to 10% to 12% SDS-PAGE and transferred to a nitrocellulose membrane (Daeillab Service Co., Ltd, Seoul, South Korea). The membranes were then blocked for 2 hr with 5% skim milk (Difco™, Sparks, MD, USA) or 5% donkey serum (Sigma-Aldrich Inc., St. Louis, MO, USA) in PBS with 0.05% Tween-20 (PBS-T). After blocking, membranes were incubated with antibodies specific for CYP19A1 (diluted 1:500) and CYP17A1 (diluted 1:500) overnight as well as HRP-conjugated anti-goat and anti-rabbit secondary antibodies (diluted 1:2000) in blocking buffer with PBS-T for 1 hr. Luminol reagent (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used to visualize binding of antibodies. Each blot was then stripped by incubation with 2% SDS and 100 mM mercaptoethanol in 62.5 mM Tris-HCl (pH 6.8) for 30 min at 50~ 60°C. Membranes were subsequently probed with antibody against β-actin (diluted 1:3000, Santa Cruz Biotechnology) as an internal control. Blots were then scanned using Gel Doc 1000, version 1.5 (Bio-Rad Laboratories Inc.), and band intensities were normalized to β-actin levels.
Gel doc 1000 version 1
Manufactured by Bio-Rad
The Gel Doc 1000, version 1.5, is a laboratory imaging system designed for the analysis of biomolecular samples, such as DNA, RNA, and proteins, separated by gel electrophoresis. The system captures and digitizes fluorescent or chemiluminescent signals from stained gels, allowing for the visualization and quantification of the separated biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Luminol reagent, by Bio-Rad (4 mentions)
Pro prep solution, by iNtRON Biotechnology (4 mentions)
Antibody against β actin, by Santa Cruz Biotechnology (2 mentions)
Nitrocellulose membranes, by Daeil Lab Service (2 mentions)
Donkey serum, by Merck Group (1 mentions)
Luminol, by Bio-Rad (1 mentions)
Elastin, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Bio-Rad (1 mentions)
Gapdh, by Abcam (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Antibody against β actin, by Cell Signaling Technology (1 mentions)
Skim milk, by Bio-Rad (1 mentions)
5 protocols using gel doc 1000 version 1
1
Western Blot Analysis of Cytochrome P450 Enzymes
2
Quantifying Skin Protein Expression
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Protein samples of NHDFs treated with essential oils were extracted using Pro‐Prep solution (iNtRON Biotechnology, Seoul, Korea), according to the manufacturer's protocol. A bicinchoninic acid assay was used to determine protein concentrations. Proteins were then loaded and separated by 8% SDS/PAGE and transferred to nitrocellulose membranes using a wet transfer system. Membranes were blocked for 2 h with 5% skimmed milk in PBS containing 0.05% Tween‐20 (PBST) at room temperature. Subsequently, membranes were incubated with antibodies against collagen 1 (1 : 500; cat. no. sc‐293 182; Santa Cruz Biotechnology, Dellas, TX, USA), collagen 3 (1 : 500; cat. no. sc‐271 249; Santa Cruz Biotechnology), elastin (1 : 1000; cat. no. ab21736; Abcam, Cambridge, UK), and GAPDH (1 : 3000; cat. no. #2118; Cell Signaling Technology, Danvers, MA, USA), which served as the internal control, overnight at 4 °C. Blots were then incubated for 1 h with horseradish peroxidase‐conjugated secondary antibodies (1 : 5000; cat. nos. ADI‐SAB‐100 and ADI‐SAB‐300; Enzo Life Science, Farmingdale, NY, USA) in 5% skimmed milk in PBST for 1 h at room temperature. Luminol (Bio‐Rad, Hercules, CA, USA) was used to visualize antibody binding. Blots were scanned using Gel Doc 1000 version 1.5 (Bio‐Rad), and protein band intensities were normalized versus GAPDH.
3
Western Blot Analysis of Steroidogenic Enzymes
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Protein samples were extracted from granulosa cells with Pro-Prep solution (iNtRON Biotechnology, Seoul, Republic of Korea) following the manufacturer's protocol. A total 30 μg of cytosolic proteins were separated by 8 to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes (Daeillab Service Co., Ltd, Seoul, Republic of Korea). The membranes were then blocked for 2 hours with 5% skim milk (DifcoTM, Sparks, MD, USA) in PBS with 0.05% Tween-20 (PBS-T). After blocking, membranes were incubated with antibodies specific for CYP11A1 (diluted 1:1,000) and 3β-HSD (diluted 1:2,000) overnight as well as HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (diluted 1:2,000) in blocking buffer with PBS-T for 1 hour. Luminol reagent (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used to visualize antibody binding. Each blot was then stripped by incubation with 2% SDS and 100 mmol/L mercaptoethanol in 62.5 mmol/L Tris-HCl (pH 6.8) for 30 minutes at 50-60°C. Membranes were subsequently probed with antibody against β-actin (diluted 1:3,000, Santa Cruz Biotechnology) as an internal control. Blots were then scanned using Gel Doc 1000, Version 1.5 (Bio-Rad), and band intensities were normalized to β-actin levels.
4
Western Blotting of Adipocyte Proteins
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For western blotting, the adipocyte protein samples were extracted using Pro-prep solution from iNtRON Biotechnology (Seoul, Korea), according to the manufacturer’s instructions. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 8–12% SDS), and transferred to nitrocellulose membranes; from Daeil Lab Service Co., Ltd (Seoul, Korea). The membranes were blocked for 2 hr with 5% Difco™ skimmed milk (Sparks, MD, USA) in PBS with 0.05% Tween-20 (PBS-T). After blocking, the membranes were incubated overnight with anti-FASN rabbit (diluted 1:1000), anti-SREBP-1c mouse (diluted 1:1000), and anti-PPARG mouse (diluted 1:1000) antibodies (Santa Cruz Biotechnology, Inc.), then further incubated for 1 hr with HRP-conjugated anti-rabbit or antimouse antibodies (diluted 1:2000) in 5% skimmed milk in PBS-T. Luminol reagent from Bio-Rad (Hercules, CA, USA) was used to visualize antibody binding. Each blot was stripped by incubation with 2% SDS and 100 mM 2-mercaptoethanol in 62.5 mM Tris-HCl (pH 6.8) for 30 min at 50–60°C. The membranes were subsequently probed with a β-actin antibody (diluted 1:3000) (Santa Cruz Biotechnology, Inc.), as an internal control. The blots were scanned using Gel Doc 1000 version 1.5 (Bio-Rad), and the band intensities were normalized to the β-actin levels.
5
Western Blot Analysis of OXTR Protein
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Protein samples were extracted with Pro-prep solution (iNtRON Biotechnology, Seoul, Korea) following the manufacturer's protocol. Cytosolic proteins (25 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Daeillab Service Co., Seoul, Korea). The membranes were then blocked for 2 h with 5% skim milk (Difco, Sparks, MD, USA) in Tris-buffered saline (TBS)with 0.05% Tween20 (TBS-T). After blocking, membranes were incubated with anti-OXTR goat (SC-8102, Santa Cruz Biotechnology, diluted 1:500) overnight, followed by HRP-conjugated anti-goat secondary antibody (SC-2020, Santa Cruz Biotechnology, diluted 1:2000) in 5% skim milk with TBS-T for 1 h. Luminol reagent (Bio-Rad) was used to visualize antibody binding. Each blot was then stripped by incubation with 2% SDS and 100 mM mercaptoethanol in 62.5 mM Tris-HCl (pH 6.8) for 30 min at 50-60°C. The membranes were subsequently probed with antibody against β-actin (#4967, Cell Signaling Technology, diluted 1:2000) as an internal control. The antibodies for OXTR (Young et al. 2014 (link)) and β-actin (Li et al. 2013) (link) were selected based on previous publication. The blots were scanned using Gel Doc 1000, version 1.5 (Bio-Rad), and band intensities were normalized to β-actin levels.
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