Cells were lysed in RIPA buffer containing a protease inhibitor cocktail (Roche, Roswell, GA, USA) on ice for 30 min; samples were then centrifuged at 12 000 × g at 4 °C for 20 min to collect the supernatants. Protein concentrations were measured using the BCA Protein Assay Kit (Thermo Scientific, USA). Samples of 50 μg were separated by 8% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, USA). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies, including rabbit anti-PTCH1 (1:1 000, Abcam, Cambridge, UK; catalogue No.: ab53715, detecting the N terminal of human PTCH1), anti-GLI1 (1:1 000, Cell Signaling Technology, Danvers, MA, USA) and mouse anti-GAPDH (1:5 000, ZSGB-BIO, Beijing, China), respectively. After three washes for 5 min each in TBST, membranes were incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA; diluted at 1:10 000) at room temperature for 1 h. Finally, the protein bands were visualized using the enhanced chemiluminescence method and an ECL detection kit (CWbiotech, Beijing, China). Band densities were analyzed by Gel-Pro Analyzer 4.0 software, and protein levels were normalized to those of GAPDH.
Horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies are a type of laboratory reagent used in immunoassays and other applications. They contain a secondary antibody that is conjugated, or linked, to the enzyme horseradish peroxidase. This allows for the detection and visualization of target proteins or other molecules when the secondary antibody binds to a primary antibody.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Luminata classico western hrp substrate, by Merck Group (4 mentions)
Immuno blot pvdf membrane, by Bio-Rad (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Las 4000, by Fujifilm (3 mentions)
Multi gauge software, by Fujifilm (3 mentions)
Cell lysis buffer, by Cell Signaling Technology (3 mentions)
Ripa buffer, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Ecl plus western blotting detection system, by Cytiva (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Clarity ecl western blotting detection reagent, by Bio-Rad (2 mentions)
Ecl kit, by CWBIO (2 mentions)
47 protocols using horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibody
1
Western Blot Analysis of Hedgehog Pathway
2
Western Blot Analysis of LATS1, YAP/TAZ, and pYAP
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GH3 cells were lysed in ice-cold RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktail (Roche). Proteins were separated by polyacrylamide gel electrophoresis and blotted using standard procedures (BioRad). Primary antibodies were against LATS1 (C66B5, #3477), YAP/TAZ (D24E4, #8418) and phosphorylated pYAP(S127) (D9W2I, #13008) (all rabbit mAb, Cell Signaling) and β-actin (mouse mAb, Chemicon). Anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies were used (Cell Signaling) and signal was developed with enhanced chemiluminescent solution (Roche). Each experiment was carried out in duplicate.
3
Protein Extraction and Western Blot Analysis in Zebrafish
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Whole-cell extracts or deyolked zebrafish larvae at 3 dpf were prepared using RIPA buffer (Sigma-Aldrich) containing 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich) and a cocktail of protease inhibitors (Roche, Basel, Switzerland) and then centrifuged at 7000g for 5 min or 12,000g for 10 min at 4°C. The protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific). An equal amount of total protein was separated on MP TGX precast gels (Bio-Rad) and transferred to polyvinylidene fluoride membranes (Bio-Rad). The membranes were blocked in tris-buffered saline (LPS Solution, Daejeon, Korea) containing 0.05% Tween 20 (TBST; Sigma-Aldrich) with 5% nonfat milk (BD Biosciences, Franklin Lakes, NJ, USA) or 1.5% BSA for 1 hour at room temperature and then incubated with specific primary antibodies overnight at 4°C. After washing with TBST six times for 30 min, the samples were incubated for 1 hour at room temperature with anti-rabbit or anti-mouse horseradish peroxidase–conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA; 1:5000). The blots were developed using ECL Select Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK). The antibodies used in this study are listed in table S3.
4
Western Blot Quantification Workflow
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Proteins separated by SDS-PAGE were transferred to polyvinylidene difluoride membrane. The membranes were blocked for 1 h at room temperature in 10 ml of blocking buffer containing 0.5% fat-free milk prior to the addition of primary polyclonal antibodies (at a dilution of 1:2000). Membranes were incubated for an additional hour, washed three times for 10 min in 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween-20, and incubated with 1:10,000 anti-rabbit or antimouse horseradish peroxidase–conjugated secondary antibody (Cell Signaling Technology) for 1 h. Membranes were washed again in 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween-20, and immunoreactive products were revealed by chemiluminescent detection using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). Blots were developed on Amersham Hyperfilm ECL (GE Healthcare Life Sciences), and band densities were quantified using ImageJ (National Institutes of Health) (54 ) software. Graphing and statistical analysis were performed using Prism 9 software (GraphPad Software, Inc).
5
Western Blot Analysis of Cardiomyocytes
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Western or immunoblotting was performed as reported previously [7] (link). Briefly, after indicated treatments, cardiomyocytes were washed and lysed. Protein concentration was determined by a bicinchoninic acid-based assay using a commercially available kit (Thermo Fisher Scientific), and 40 µg of protein was loaded onto 10 or 12% SDS-PAGE gel for electrophoresis. Proteins were transferred to polyvinylidene fluoride (PVDF) membrane, which was blocked with 5% milk and probed with primary antibody against the protein of interest, then with anti-rabbit or anti-mouse horseradish peroxidase conjugated secondary antibody (#7074, Cell Signaling Technology) in a standard manner. Primary antibodies used for western blotting include polyclonal antibody against actin (#4968), Cyt c (#4272), ATF-2 (#9226), and CoxIV (#4844) from Cell Signaling Technology; SOD-2 (#sc-137254), PGM-1 (#sc-50656), phosphorylated p38 (#sc-17852-R), and p38β (#sc-6187-R) from Santa Cruz Biotechnology. The membrane was then incubated with chemiluminescence reagents using a commercially available ECL Plus Western Blotting Detection System (Amersham Biosciences) and exposed to film. The band intensity on radiographs was determined by densitometry and NIH ImageJ.
6
Nuclear Protein Extraction and Western Blotting Analysis
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The nuclear protein extracts were prepared from HaCaT cells using NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Rockford, IL), according to the manufacturer’s protocol. Additionally, total protein was extracted by ice-cold RIPA buffer (Nacalai Tesque) with protease inhibitor cocktail. The protein samples were subsequently prepared 4× Laemmli Sample Buffer (Bio-Rad Laboratories, Forester City, CA) and boiled at 100°C for 5 min. Equal amounts of protein were separated by SDS-PAGE and then transferred onto polyvinylidene difluoride membranes for 35 min at 100 V. After that, the membranes were blocked with Blocking One (Nacalai Tesque) for 40 min at room temperature. The membranes were incubated at 4°C overnight in each primary antibody. Primary antibodies specific to SIRT-1, HO-1, NF-κB, histone H3 and β-actin (Cell Signaling Technology) and Nrf2 were used. Membranes were washed by TBST three times and incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology). To detect the immunocomplex, an Immobilon HRP substrate (Bio-Rad Laboratories) was used. Immunoblots were scanned by densitometry and the intensity was quantified by using Image Lab software (Bio-Rad Laboratories). The relative levels of SIRT-1 and HO-1 were normalized to β-actin, and nuclear Nrf2 and NF-κB were standardized to histone H3.
7
Protein Expression Analysis by Western Blot
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Tissue lysates were extracted using RiPA Lysis buffer (Millipore, Burlington, MA, USA). Equal amounts of protein (20–40 μg) were loaded and separated using a 10% SDS-PAGE gel and electrophoretically transferred onto polyvinylidene difluoride microporous membranes (0.45 μm pore size, Millipore, Sigma-Aldrich, St. Louis, MO, USA). Membranes were blocked using TBS with Tween 20 (TBS-T) containing 5% bovine serum albumin (1 h, room temperature) and incubated with anti- vascular endothelial growth factor (VEGF)-A (1:1000; Santa Cruz, Dallas, TX, USA) or HSP-90 (1: 10,000, Santa Cruz, Dallas, TX, USA) primary antibodies overnight (4 °C) on a shaker. After the membranes were washed with TBS-T and incubated with either anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody (Cell Signaling, Beverly, MA, USA) (1 h, room temperature), immunocomplexes were visualized using chemiluminescence (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s instructions.
8
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Triton X‐100 lysis buffer [20 mm Tris (pH 7.4), 2 mm EDTA, 150 mm sodium chloride, 1 mm sodium deoxycholate, 1% Triton X‐100, 10% glycerol, 2 pills protease inhibitor cocktail (Roche)] was used to collect protein from cells. Protein samples were heat‐denatured and equally loaded, separated on 8–12% SDS/PAGE gel, transferred onto a polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, UK), and blocked with 5% nonfat dry milk. Primary antibodies for western blot analyses included mouse anti‐α‐tubulin (1 : 20 000 dilution; Sigma), rabbit anti‐Snail (1 : 1000 dilution; Cell Signaling, Danvers, MA, USA), rabbit anti‐Slug (1 : 1000 dilution; Cell Signaling), rabbit anti‐CHIP (1 : 1000 dilution; Abgent, San Diego, CA, USA), mouse anti‐Flag (1 : 3000 dilution; Abcam), mouse anti‐GFP (1 : 1000 dilution; Santa Cruz), mouse anti‐HA (1 : 1000 dilution; Abcam, Cambridge, MA, USA), mouse anti‐Vimentin (1 : 1000 dilution; Santa Cruz, Santa Cruz, CA, USA), mouse anti‐E‐cadherin (1 : 5000 dilution; BD Biosciences, San Jose, CA, USA), and mouse anti‐Ub (1 : 5000 dilution; Santa Cruz). Membranes were incubated with horseradish peroxidase‐conjugated anti‐mouse or anti‐rabbit secondary antibody (1 : 5000 dilution; Cell Signaling) for 1 h, and chemiluminescence signals were detected by ECL substrate (GE Healthcare, Chicago, IL, USA).
9
Western Blot Protein Analysis
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Cells (2 × 106) were lysed in RIPA lysis buffer (Beyotime Biotechnology) and quantitated using a BCA Protein Assay Kit (Beyotime Biotechnology). The protein liquid was mixed with 5 × loading buffer in a volume ratio of 4 to 1, and placed in a boiling water bath for 10 min to denature. For Western blot analysis, equivalent amounts of protein per sample were electrophoretically resolved on 10% polyacrylamide gels and then transferred onto PVDF membranes (Millipore). Following this, the PVDF membranes with the protein were blocked with blocking buffer (Beyotime Biotechnology), then incubated with the corresponding primary antibodies. After repeated washes, the membranes were incubated with horseradish‐peroxidase‐conjugated anti‐mouse or anti‐rabbit secondary antibody (Cell Signaling, 1:1000 diluted) at room temperature for 1 h. An electro‐chemiluminescence (ECL) system (Thermo Fisher Scientific) was used for the detection. Anti‐β‐actin (Epitomics) was used to check for equal loading of protein between wells. The Primary antibodies used for the Western blot are shown in Table 2 .
10
Apoptosis Pathways in A549 Cells
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The experimental groups were divided into control group, chitin oligosaccharide group, cisplatin group and chitin oligosaccharide plus cisplatin group. A549 cells were collected after incubated for 72 hrs and lysed with RIPA buffer. The concentrations of proteins were measured by BCA method and samples were separated by SDS-PAGE. After transfer, the PVDF membrane was blocked with 5% fat-free milk for 2 hrs and incubated with primary antibodies at 4°C overnight. Primary antibodies were rabbit anti-human caspase3 antibody (1:500; cell signaling technology, USA), mouse anti-human caspase8 antibody (1:500; cell signaling technology, USA), rabbit anti-human BAK antibody (1:500; cell signaling technology, USA) and mouse anti-human β-actin antibody (1:500; santa cruz biotechnology, USA). The membrane was then incubated with a horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (1:40,000; cell signaling technology, USA) after it was rinsed by TBS-T for three times. ECL method was used for immunoblot analysis.
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