Samples from liver tissues homogenate and cell lysates were separated by SDS‐PAGE, and transferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes were incubated with primary antibodies, and then incubated with HRP‐conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA). Anti‐MMP21 (55289‐1‐AP), anti‐Flag‐tag (20543‐1‐AP), anti‐CCL14 (14216‐1‐AP), and anti‐GAPDH (60004‐1‐IG) antibodies were purchased from ProteinTech. Anti‐ZEB2 (#97885), anti‐Twist (#69366) and the Epithelial–Mesenchymal Transition (EMT) antibody sampler kit (#9782T) including anti‐vimentin, anti‐Snail, anti‐β‐catenin, anti‐E‐cadherin, anti‐N‐cadherin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The images were visualized using an enhanced chemiluminescence detection kit (Tiangen Biotech, Beijing, China) and the ImageQuant™ LAS‐4000 system (Amersham Biosciences, GE, USA).
Enhanced chemiluminescence detection kit
Manufactured by Tiangen Biotech
Sourced in China
The Enhanced chemiluminescence detection kit is a laboratory equipment product that enables detection and visualization of proteins or other biomolecules using chemiluminescence technology. The kit contains the necessary reagents and materials for performing chemiluminescent-based Western blot analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Complete protease inhibitor, by Roche (4 mentions)
Imagequant las 4000, by GE Healthcare (3 mentions)
Las 4000, by Cytiva (3 mentions)
Quantity one v4, by Bio-Rad (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Phosstop phosphastase inhibitors, by Roche (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Protein g ip kit, by Roche (2 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Anti n cadherin, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti gapdh 60004 1 ig, by Proteintech (1 mentions)
Imagequant las 4000 system, by Cytiva (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Emt antibody sampler kit, by Cell Signaling Technology (1 mentions)
Anti snail, by Cell Signaling Technology (1 mentions)
12 protocols using enhanced chemiluminescence detection kit
1
Protein Expression Analysis by Western Blot
2
Western Blot Analysis of EGFL7 and HIF-1α
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Protein samples were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Polyvinylidene Fluoride (PVDF) membranes. After blocking, the membranes were incubated with the indicated specific primary Abs for 2 h at room temperature followed by washing and incubation with horseradish peroxidase (HRP)-conjugated secondary Ab. The involved Abs were rabbit polyclonal anti-EGFL7 and anti-HIF-1α Abs (1:1000, Abcam, Cambridge, MA, USA) and rabbit monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2000, Abcam, Cambridge, MA, USA). The secondary Ab was an HRP-conjugated anti-rabbit IgG Ab (1:4000; Zhongshan, Beijing, China). The immunoreactive proteins were developed with enhanced chemiluminescence detection kit (Tiangen Biotech) and visualized with the image analyzer Image Quant LAS 4000 (GE Healthcare).
3
Western Blot Protein Analysis
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Cell pellets were solubilized with the RIPA buffer (Beyotime Institute of Biotechnology, China) with the addition of cOmplete Protease Inhibitors (Roche, Switzerland) and PhosSTOP Phosphastase Inhibitors (Roche, Switzerland), electrophoresed, and blotted onto polyvinylidene fluoride membranes. The membranes were incubated with the indicated primary antibodies, followed by the incubation with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, UK). The protein concentration was determined with the BCA Protein assay kit (Thermo Scientific Pierce, UK). Blotted proteins were visualized using an enhanced chemiluminescence detection kit (Tiangen Biotech, China). The intensity of the bands was analyzed by Quantity One V 4.62 software (Bio-Rad, Life Science, CA, USA) [3 (link)].
4
Quantitative Western Blot Analysis
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Western blot analysis was carried out as previously described (25 (link)). Briefly, tissues and cell lysates were collected, and the protein concentrations were determined using a BCA™ Protein Assay kit (Pierce, Chemical Co., Rockford, IL, USA). Proteins were separated by 10% SDS-PAGE and electro-transferred onto PVDF membranes (Millipore). The membranes were first incubated with the indicated primary antibodies overnight (4°C), followed by HRP-conjugated secondary antibodies for 2 h (room temperature). Blots were visualized using an enhanced chemiluminescence detection kit (Tiangen Biotech Co., Ltd. Beijing, China). The following antibodies were used: mouse monoclonal Kindlin-1 antibody (cat. no. SAB4200465; 1:1,000, Sigma), rabbit monoclonal integrin β1 antibody (cat. no. NB110-57123; 1:1,000, Novus Biologicals, Littleton, CO, USA), mouse monoclonal activated integrin β1 antibody (cat. no. MAB2079Z; 1:1,000, Millipore), rabbit polyclonal FAK antibody (cat. no. 3285; 1:1,000, Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit monoclonal Phospho-FAK (Tyr397) antibody (cat. no. 8556; 1:1,000, Cell Signaling Technology, Inc.), rabbit monoclonal GAPDH antibody (cat. no. 3683, 1:1,000; Cell Signaling Technology, Inc.). All experiments were repeated at least 4 times.
5
Western Blot Analysis of EMT Markers
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Protein in cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Hessen state, Germany), followed by incubated with primary antibodies against CLEC3B (Abcam, Massachusetts, US), HIF-1α, VEGF (Proteintech, Chicago, US), phospho-AMPKα, AMPKα, CD31, CD34 and EMT associated molecules (Cell Signaling Technology, Danvers, US) overnight in 4 °C. Next day, the membranes were subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, California, US). The immunoreactive protein were visualized by using enhanced chemiluminescence detection kit (Tiangen Biotech, Beijing, China) and image analyzer ImageQuant LAS 4000 (GE Healthcare, Abingdon, UK). To ensure equal loading of plasma protein, the membranes were stained with 0.2% Ponceau S (Sinopharm Chemical Reagent, Shanghai, China) to ensure equal loading of the proteins.
6
Western Blot Analysis of ANXA3 Protein
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Proteins were extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology), then cells were centrifuged at 12,000 × g for 20 min at 4°C. Supernatants containing 20–30 µg protein were collected, and the protein content was quantified using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Samples (20 µg/lane) were separated by 12% SDS-PAGE and transferred to PVDF membranes. The blots were blocked with 5% skim milk in TBST for 2 h at room temperature, incubated with primary anti-ANXA3 (1:1,000) or anti-β-actin (1:2,000) primary antibodies at 4°C overnight. After washing with TBST (0.05% Tween-20), the membranes were incubated with HRP-conjugated anti-rabbit secondary antibodies (1:5,000) or HRP-conjugated anti-mouse secondary antibodies (1:5,000) at 27°C for 2 h. The bands were visualized using an enhanced chemiluminescence detection kit (Tiangen Biotech Co., Ltd.) and quantified by Quantity One software (version 2.4; Bio-Rad Laboratories, Inc.).
7
Western Blot Protein Detection
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Protein from cell lysates were separated by SDS–polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes (Millipore), and incubated with primary antibodies, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology). The immunoreactive proteins were visualized by using enhanced chemiluminescence detection kit (Tiangen Biotech, Beijing, China) and the image analyzer ImageQuant LAS 4000 (GE Healthcare, Abingdon, UK).
8
Quantification of Osteogenic Markers
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Each culture was rinsed twice with PBS and solubilized with radio immunoprecipitation assay (RIPA) buffer. The protein concentration was determined with the Micro BCA Protein Assay Reagent Kit (Kangchen Bio-tech Company, Shanghai, China). Approximately 35 μg of proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to determine the levels of COLⅠ, RUNX2, OCN and β-catenin. Subsequently, the proteins were transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA). The blots were blocked by incubation in 5% milk with TBST for 1 h and probed overnight at 4 ℃ with the rabbit anti-COL I, rabbit anti-RUNX2, rabbit anti-OCN and rabbit anti-β-catenin antibodies, respectively. After washing, the membranes were incubated with the appropriate HRP-conjugated secondary antibody (1:5000 in 5% milk) for 60 min at room temperature. The immunoreactive bands were visualized using an enhanced chemiluminescence detection kit (TIANGEN BIOTECH CO., LTD., Beijing, China). The optical density of the protein bands was determined using a Gel Doc 2000 (Bio-Rad, CA, USA). The expression of GAPDH was used as a loading control, and the data were normalized against the corresponding GAPDH band. The results were expressed relative to the control.
9
Western Blot Analysis of Adipose Proteins
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Total proteins were isolated from white adipose tissues using RIPA (Solarbio Life Science, Beijing, China) buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined using a protein assay reagent (Beyotime Biotechnology, China) and equal amounts of protein (60 μg) were loaded in each well of an 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. After the proteins were transferred onto a polyvinylidene difluoride membranes, the blots were blocked with 5% bovine serum albumin, followed by incubation overnight at 4°C with the following primary antibodies: anti-AMPKα (23A3), anti-phospho-AMPKα (Thr172), anti-ACC, anti-FAS, and anti-CPT1α (Cell Signaling Technology, United States). After three washes with TBST buffer, the blots were incubated with appropriately diluted horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The immunoblots were visualized with an enhanced chemiluminescence detection kit (Tiangen Biotech, Beijing, China). Protein levels were normalized to tubulin as a loading control.
10
Western Blot Analysis of Protein Expression
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Cell pellets were solubilized with RIPA buffer (Radio-Immunoprecipitation Assay, Beyotime Biotech, CHINA) with the addition of complete Protease Inhibitors (Roche, Switzerland) and Phosphatase Inhibitors (Roche, Switzerland), electrophoresed, and blotted onto PVDF (Polyvinylidene Fluoride) membranes. The membranes were incubated with indicated primary antibodies followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Beyotime Biotech, CHINA). Protein concentration was calculated by the BCA (Bicinchoninic Acid) Protein assay kit (Thermo Scientific Pierce, UK). Blotted proteins were visualized using an enhanced chemiluminescence detection kit (Tiangen Biotech, CHINA). The intensity of the bands was analysed by Quantity One V 4.62 Software (Bio-Rad, Life Science, USA). The relative protein expression was calculated by Image J software using the formula”Gray value of targeted protein band/Gray value of interior reference band (such as β-actin, COX4, etc)”.
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