Total protein was extracted using RIPA buffer, and the protein concentration was determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein (30 µg) of each sample was loaded onto a 10% SDS-PAGE gel, resolved, then transferred to a polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA). After blocking with 5% bovine serum albumin at room temperature for 1 h, the membranes were incubated with primary antibodies (1:1,000 dilution) at 4 °C overnight. After the membranes were incubated with the corresponding secondary antibodies (1:5,000 dilution) for 1 h at room temperature, the blots were detected using an enhanced chemiluminescence solution (Invitrogen) and visualized with a ImageQuantLAS-4010 (GE Healthcare, Chicago, IL, USA).
Enhanced chemiluminescence solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
Enhanced chemiluminescence solution is a reagent used in Western blot analysis. It is designed to produce a luminescent signal when in contact with the target protein, enabling the detection and visualization of the protein of interest.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (12 mentions)
Las 3000, by Fujifilm (6 mentions)
β actin, by Merck Group (6 mentions)
Las 4000, by Cytiva (5 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (5 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
Anti mouse or anti rabbit horseradish peroxidase conjugated antibodies, by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
β actin, by Bioworld Technology (3 mentions)
Sirt1, by Santa Cruz Biotechnology (3 mentions)
Protease inhibitor, by Roche (3 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Imagequant las 4010, by GE Healthcare (2 mentions)
Gapdh, by Abcam (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Histone h3, by Bioworld Technology (2 mentions)
91 protocols using enhanced chemiluminescence solution
1
Western Blot Protein Detection
2
Western Blot Protein Analysis
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Equal amounts of protein (30 μg) were subjected to electrophoretic separation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (SDS-PAGE) and subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, USA). After blocking with nonfat milk in PBST for 2 h at room temperature, the PVDF membranes were incubated with primary antibodies at 4°C overnight. Information about these antibodies is shown in Table 3. After washing with PBST, the membranes were incubated with secondary antibodies at room temperature for 2 h. The membranes were developed with an enhanced chemiluminescence solution (Invitrogen) and analyzed using Image Lab 5.1 software.
3
Western Blot Protein Detection Protocol
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Western blot assays were conducted as previously described (Yang et al., 2016 ). Protein samples were separated using SDS‐PAGE and transferred onto nitrocellulose membranes. Blots were blocked using phosphate‐buffered saline (PBS) containing 5% skimmed milk, rinsed several times with PBS, and then probed with a specific primary mouse antibody (at a 1:5,000 dilution) followed by an anti‐mouse (at a 1:10,000 dilution) alkaline phosphatase conjugate. The detection signal was visualized by incubating blots in an enhanced chemiluminescence solution according to the manufacturer's instructions (Invitrogen) and scanned using an Amersham Imager 680 machine (GE Healthcare BioSciences).
4
Western Blot Analysis of Protein Samples
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The total protein was extracted using RIPA buffer. The protein concentration was quanti ed by the BCA Protein Assay kit (Thermo). Protein (30 µg) of each sample was loaded into a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel(SDS-PAGE gels) and transferred onto polyvinylidene uoride (PVDF) membranes (Millipore). After blocking with 5% BSA at room temperature for 1 h, membranes were incubated with primary antibodies (1/1000 dilution) at 4℃ overnight. After membranes were incubated with corresponding secondary antibodies (1/5000 dilution) for 1 h at room temperature. Blots were detected using enhanced chemiluminescence solution (Invitrogen) and visualized with the ImageQuantLAS-4010 (GE).
5
Protein Expression Analysis by Western Blotting
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Western blotting was performed as previously described.18 Briefly, cells were harvested, lysed, and centrifuged. Equal amounts of proteins were separated by SDS‐PAGE and transferred onto PVDF membranes (Millipore). The membranes were blocked in 5% nonfat milk and incubated with primary antibodies for TGF‐β1, SPHK1, E‐cadherin, N‐cadherin, α‐SMA, and GAPDH (Abcam) at 4°C overnight. After incubation in the corresponding secondary antibodies, membranes were incubated in the enhanced chemiluminescence solution (Thermo Fisher Scientific).
6
TGF-β1 Induced Protein Expression
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LX-2 cells were treated with 10 ng/mL TGF-β1 for the indicated times, followed by a harvest step using RIPA buffer. The extracted protein lysates were normalized using the BCA protein assay kit (Thermo Fisher Scientific). Normalized protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Merck KGaA, Darmstadt, Germany). The membranes were blocked with 3% skim milk in Tris-buffered saline containing 0.1% Tween 20, probed with primary antibodies overnight at 4 °C, and then immunoblotted with the corresponding secondary antibodies (see below) for 4 h at room temperature. The membranes were then exposed to an enhanced chemiluminescence solution (Thermo Fisher Scientific) and visualized using an Amersham ImageQuant 800 (Marlborough, MA, USA). The protein band of Western blotting was quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).
7
SARS-CoV-2 N Protein Detection by Western Blot
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SARS-CoV-2 particles were lysed with a lysis buffer containing 10 mM HEPES, 150 mM NaCl, 5 mM EDTA, 100 mM NaF, 2 mM Na3VO4, protease inhibitor cocktail, and 1% NP-40. Each virus lysate was loaded on a 4–12% Bis-Tris gradient gel (Thermo Fisher Scientific) and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk solution in phosphate-buffered saline containing 0.1% Tween-20 (PBST) and incubated with the mouse anti-SARS-CoV-2 N protein mAb (2A7H9 clone or 1G10C4 clone) and then with horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG secondary antibody (Catalog No. 715-035-150, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Enhanced chemiluminescence solution (Thermo Fisher Scientific) was used to detect immune-reactive proteins.
8
SDS-PAGE and Western Blot Protocol
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48h post-transfection, cells were lysed in RIPA-buffer supplemented with α-complete protease inhibitor cocktail (La Roche), sonicated and protein concentrations were determined using BCA total protein determination kit (Thermo Fisher Scientific). Equal amounts of protein were resolved by SDS-PAGE analysis using 10% gels. After electrophoresis, proteins were transferred to nitrocellulose membranes (Bio-Rad) and subsequently blocked for 1h at 22°C in 5% non-fat milk in 0.1% Tween-20/TBS solution. Then, membranes were incubated overnight at 4°C with indicated primary antibody in 5% BSA in 0.1% Tween-20/TBS solution. The next day, membranes were incubated with HRP-conjugated secondary antibody in blocking buffer for 1h at 22°C. Immunoblots were developed using enhanced chemiluminescence solution (Thermo Fisher Scientific).
9
Quantification of Skin and Cell Proteins
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Total protein from skin tissue and BJ cells was extracted using RIPA buffer (Beyotime Institute of Biotechnology) and then quantified using a Pierce™ BCA Protein assay kit (cat. no. 23225; Thermo Fisher Scientific, Inc.). A total of 40 µg protein/lane was separated via SDS-PAGE (Mini-Protean-3; Bio-Rad Laboratories, Inc.) and transferred to polyvinylidene difluoride membranes (EMD Millipore). The membrane was blocked with 5% skimmed milk powder solution for 1 h and then incubated with the following primary antibodies at 4˚C overnight: Rabbit anti-collagen I antibody (cat. no. ab34710; 1:2,000 dilution; Abcam), rabbit anti-collagen III antibody (cat. no. ab7778; 1:5,000 dilution; Abcam), rabbit anti-MMP-1 antibody (cat. no. ab137332; 1:1,000 dilution; Abcam), rabbit anti-α-SMA antibody (cat. no. YM-H0645; 1:500 dilution; Shanghai Yuan Mu Biotechnology Co., Ltd.) and rabbit anti-β-actin antibody (cat. no. ab8227; 1:2,00 dilution; Abcam). The protein bands were then incubated with secondary antibody goat anti-rabbit IgG (cat. no. ab6721; 1:2,000 dilution; Abcam) at room temperature for 2 h and treated with enhanced chemiluminescence solution (Thermo Fisher Scientific, Inc.). β-actin was used as the internal reference and the gray values of protein bands were quantitatively analyzed by ImageJ software (version 1.46r; National Institutes of Health).
10
Protein Immunoblot Analysis of Liver Tissue
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For protein immunoblot analysis, mouse liver tissue lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred to polyvinylidene difluoride membranes (L-IPVH 00010; Merck Millipore, Burlington, Massachusetts, USA). The membranes were incubated with antibodies against smooth muscle actin (SMA; ab5694; Abcam, Cambridge, England, UK), cleaved Poly (ADP-ribose) polymerase (c-PARP; 9544S; Cell Signaling Technology, Danvers, Massachusetts, USA), cleaved caspase 3 (c-Caspase 3; 9661S; Cell Signaling Technology), and β-actin (sc-47778; Santa Cruz Biotechnology, Dallas, Texas, USA) at 4°C overnight. Subsequently, the membranes were incubated with secondary antibodies at room temperature for 1 h. The blots were developed using an enhanced chemiluminescence solution (#34580; Thermo Fisher Scientific, Waltham, Massachusetts, USA).
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