Cells were lysed with 1% SDS. Samples were boiled in boiling water for 20 min and centrifuged at 12,000 rpm for 10 min at 4 °C to remove cell debris. The total protein content of the supernatants was measured using a BCA protein assay kit (ThermoFisher Scientific, Shanghai, China) and was mixed with 5× loading sample buffer (Biosharp, BL502A, Hefei, China). Then, samples were boiled in boiling water for 10 min. Equal amounts of proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes (NC membrane, Millipore, Wuhan, China). NC membranes were incubated with 5% nonfat milk for 2 h at room temperature to block nonspecific proteins. Then, NC membranes were incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies for 2 h at room temperature. Antibodies were used following the instructions. Later, NC membranes were detected by chemiluminescence using a WesternBrightTM ECL Western blotting detection kit (Advansta, Menlo Park, USA).
Westernbright ecl western blotting detection kit
Manufactured by Advansta
Sourced in United States
WesternBright ECL is a chemiluminescent western blotting detection kit. It is used to detect and quantify proteins on western blots.
Automatically generated - may contain errors
Lab products found in correlation
Nc membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Bl502a, by Biosharp (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ovalbumin (ova), by InvivoGen (1 mentions)
Bovine serum albumin fraction 5, by Roche (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Du145, by American Type Culture Collection (1 mentions)
Anti mouse igg 7076, by Cell Signaling Technology (1 mentions)
Quick start bradford 1x dye reagent, by Bio-Rad (1 mentions)
Complete edta free protease inhibitor cocktail tablet, by Roche (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Lncap, by American Type Culture Collection (1 mentions)
Cwr22rv1, by American Type Culture Collection (1 mentions)
Ab16801, by Abcam (1 mentions)
10 protocols using westernbright ecl western blotting detection kit
1
Western Blot Protein Analysis
2
Western Blot Analysis of NDRG1 Expression
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Proteins were extracted from 16 pairs of samples by RIPA lysis buffer, segregated by 12% SDS-PAGE, and then transferred to PVDF membranes (Millipore, USA, IPVH00010). Skim milk powder was dissolved in TBST, and membranes were blocked in 5% skim milk for 2 h, and incubated with rabbit anti-NDRG1 (#9485, CST) or rabbit anti-GAPDH (AB-P-R 001, Hangzhou Goodhere Biotechnology Co.,). The PVDF membranes were washed for 3 times, and then incubated with anti-rabbit antibody with horseradish peroxidase (HRP, #7074S, CST). Protein Bands were captured with C300 (Azure Biosystems, Azure biosystem Inc, USA) by Western BrightTM ECL western blotting detection kit (Advansta, USA). The gray values of protein bands were measured by Image J software (v1.8.0), with GAPDH as an internal control.
3
Immunoprecipitation and Western Blot Analysis
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Treated and un-treated cells were collected, and the cell extracts were prepared by incubating the cells in NHG buffer (1% Triton X-100, 10% glycerol, 50 mM HEPES, pH 7.2, and 150 mM NaCl) on ice for 30 min with protease and phosphatase inhibitors (MCE). Cell debris was removed by centrifuging the cell extracts for 15 min at 12000 rpm, and the concentration of total protein was measured using the Pierce™ BCA Protein Assay Kit (Invitrogen). The cleared cell lysates were mixed with specific antibodies for 1 h at room temperature and then coincubated with Dynabeads® Protein G at 4 °C overnight. Nonspecifically bound proteins were removed by washing the beads five times with washing buffer (1% Triton X-100, 10% glycerol, 50 mM HEPES, pH 7.2, and 400 mM NaCl). Equal amounts of proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (NC membrane, Millipore). The membrane was blocked by incubation with 5% skim milk for 2 h at room temperature. Then, the NC membrane was incubated with the primary antibody at 4 °C overnight, which was followed by incubation with the secondary antibody. Subsequently, the proteins were detected by chemiluminescence using a WesternBrightTM ECL western blotting detection kit (Advansta).
4
Soluble RAGE Receptor Quantification in E. coli
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Soluble AGE product-specific receptor human E. coli (RD172116100) was obtained from Biovendor (Brno, Czech Republic). Anti-RAGE antibody (monoclonal mouse IgG2B clone, MAB11451) was purchased from R&D systems (Minneapolis, MN, USA). Horse radish peroxidase (HRP)-conjugated anti-mouse polyclonal goat (P0447) was purchased from Dako (Glostrup, Denmark). 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate for high sensitivity ELISA was purchased from SDT reagents (Baesweiler, Germany). WesternBrightTM ECL Western blotting detection kit was obtained from Advansta (San Jose, CA, USA). Ovalbumin was purchased from InvivoGen (San Diego, CA, USA). Bovine serum albumin fraction V was obtained from Roche (Basel, Switzerland). Amyloid-β (1-42) ultrapure HFIP was purchased from Westburg (Leusden, The Netherlands). Raw bulk milk was obtained from the University Farm of Wageningen University (Wageningen, The Netherlands).
5
Western Blot Analysis of Proteins
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MV4-11 cells were collected and total protein was lysed in cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris [pH8], 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS), containing protease and phosphatase inhibitors (Bayotime, Shanghai, China). In certain experiment, nuclear and cytoplasmic protein was extracted using Nuclear and Cytoplasmic Protein Extraction Kit (Bayotime, Shanghai, China). Protein samples were resolved by 10% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (PVDF, Millipore Corporation, MA). PVDF membranes were blocked in Tris-buffered saline (TBS; 25 mM Tris, 0.15 M NaCl; pH 7.2) containing 5% nonfat dry milk and 0.1% Tween-20 for 1 hour and then incubated overnight in 4°C with various primary antibodies. After washing with TBST (TBS with 0.1% Tween-20), blots were incubated with horseradish peroxidase-linked secondary antibody at room temperature for 1 hour. Blots were developed using WesternBright ECL western blotting detection kit (Advansta Inc., Menlo Park, CA).
6
Western Blot Protein Analysis Workflow
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Fresh samples were lysed with RIPA lysis buffer and the proteins were quantified using the BCA Protein Assay Kit (Beyotime, China). Equal volumes of the proteins were then separated by 10% SDS-PAGE and transferred onto PVDF membranes. To prevent nonspecific antigen sites, 5% skim milk was applied for 1 h and the primary antibody was then incubated overnight at 4 °C. The membranes that had been washed three times were then incubated for 2 h with a 1:5000 dilution of anti-rabbit antibody with horseradish peroxidase (#7074S; CST, USA). C300 (Azure Biosystems, USA) was used to capture protein bands, which were then detected and measured using Image J software (v1.8.0) with the assistance of Western Bright™ ECL western blotting detection kit (K-12045-D50; Advansta, USA).
7
Profiling Prostate Cancer Cell Lines
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Human prostate cancer cell lines (LNCaP, VCaP, CWR22Rv1 (referred to as 22Rv1), PC3, and Du145) were purchased from American Type Culture Collection (Manassas, VA, USA). RPMI 1640 media, phenol red-free RPMI 1640 media, Fetal Bovine Serum (FBS), Charcoal Stripped FBS One Shot, 1X phosphate buffered saline (PBS) and 100X Antibiotic-Antimycotic (A/A) solutions were purchased from Gibco-Invitrogen Corporation (Carlsbad, CA). Quick Start Bradford 1X dye reagent, 4X Laemmli sample buffer, a Mini Trans-Blot wet transfer system PVDF membranes and non-fat dry milk blocking grade blocker were purchased from Bio-Rad Laboratories (Hercules, CA). cOmplete, EDTA-free Protease Inhibitor Cocktail Tablets were purchased from Roche Diagnostics (Indianapolis, IN). PARP (9542), pAKT (2920), AKT (4691), pS6 (4858) S6 (2317) primary antibodies and secondary anti-mouse IgG (7076) and anti-rabbit (7074) horseradish peroxidase (HRP)-conjugated antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, Massachusetts). Western Bright ECL western blotting detection kit was purchased from Advansta (Menlo Park, CA).
8
Western Blot Analysis of Oxidative Stress Proteins
9
Western Blotting Analysis of Protein Expression
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A standard western blotting protocol was used as described previously [50 (link)]. Briefly, control and virus-infected cells were lysed in cell lysis buffer. The concentration of total protein was determined using a BCA Protein Assay Reagent kit (SuperSignal West Dura Trial kit, Pierce, Covance, USA). Equal amounts (40–60 μg) of protein were electrophoresed, transferred to nitrocellulose membranes, and incubated with the primary antibody. Peroxidase-conjugated secondary antibody and a WesternBright ECL western blotting detection kit (Advansta, Menlo Park, CA, USA) were used to detect the signals with the Western Blotting System and analyzed with the Azure c400 Imaging System (Azure Biosystems, USA). In addition, subcellular fractionation experiments were used to estimate the expression of the molecules in the nuclear fraction including lipin 1 and sterol regulatory element-binding protein (SREBP). Nucleoproteins were separated from cells using the nuclear and cytoplasmic protein extraction kit (Beyotime, Shanghai, China) (specific procedures are shown in the Supplementary information ). CDKA and histone 3 (H3) were used as the loading controls. The primary antibodies used are shown in Supplementary Table S3 .
10
ITGA2 Protein Expression Analysis
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The cells were lysed in lysis buffer (5% glycerol, 1 mM sodium EDTA, 1 mM dithiothreitol, 40 μg/ml leupeptin, 40 μg/ml aprotinin, 20 μg/ml pepstatin, 1 mM PMSF, 0.5% Triton X-100 in PBS) and then incubated on ice for 30 min. After centrifugation at 10,000 xg for 30 min at 4 °C, supernatants were collected and the protein concentrations were determined by Bradford protein assay (Bio-Rad, Hercules, CA, USA). Fifty μg of the protein lysates were resolved by 10% SDS-PAGE. The resolved proteins were transferred onto 0.2 μm PVDF membranes (PALL). After blocking the membranes with 5% skim milk in TBST buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20), a mouse anti-human ITGA2 monoclonal antibody (1:1000 dilution; Santa Cruz Biotechnology, Paso Robles, CA, USA) was added to the membranes in TBST supplemented with 5% skim milk at 4 °C overnight or at room temperature for 1 h. After extensive washing in TBST, the membranes were then probed with a goat anti-mouse peroxidase-conjugated antibody (1:10,000 dilution; Sigma–Aldrich) at room temperature for 1 h and then washed in TBST. Finally, the bound proteins were revealed by the WesternBright ECL Western blotting detection kit (Advansta, Menlo Park, CA, USA) and imaged by a CCD camera.
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