Total proteins in differentiated C2C12 cells were extracted using RIPA buffer and quantified with BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Proteins in the sample buffer were boiled for 10 min and resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (ATTO Corp., Tokyo, Japan) at 4 °C for 80 min at 120 mA. After blocking with TBS-T (10 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.6) containing 5% bovine serum albumin (BSA) for 1 h, the membranes were then incubated with a 1:1000 dilution of primary antibodies targeting the following; phospho-AKT (S473), AKT, and β-actin. After removing primary antibodies, the blots were washed three times in TBS-T, incubated with anti-rabbit antibody diluted 1:2000 in TBS containing 5% BSA for 5 h at 4 °C and then washed five times in TBS-T. Antibody-bound proteins were detected using enhanced chemiluminescence (AbFrontier, Seoul, Korea) and imaging systems (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instruction. The expression level of each protein was normalized to β-actin and the intensities of the bands were quantified using Quantity One (Bio-Rad, Hercules, CA, USA) [38 , 39 ].
Imaging systems
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
Bio-Rad Imaging Systems are high-performance imaging solutions designed for a variety of applications in molecular biology, protein research, and life science laboratories. These systems offer advanced image capture, analysis, and documentation capabilities.
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Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ecl kit, by Merck Group (2 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by ATTO Corporation (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Cold lysis buffer, by Solarbio (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Horseradish peroxidase conjugated igg, by Thermo Fisher Scientific (1 mentions)
Quantity one image software, by Bio-Rad (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
β actin 8457, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Keygen Biotech (1 mentions)
Ecl detection system, by Thermo Fisher Scientific (1 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
10 protocols using imaging systems
1
Protein Quantification and Western Blot Analysis in Differentiated C2C12 Cells
2
Liver Protein Extraction and Western Blot Analysis
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The liver samples were lysed with cold lysis buffer (Solarbio, Beijing, China), and the samples homogenates were centrifuged at 13,000 rpm for 15 min at 4 °C. The protein concentrations of supernatant were determined with a BCA protein assay kit (Beyotime, Jiangsu, China). The protein samples were mixed with loading buffer in a volume ratio of 4:1 and then boiled for 10 min. Subsequently, the protein samples were separated on 5% SDS-PAGE and transferred to a polyvinylidene fl uoride (PVDF) membrane . The PVDF membranes were blocked for 1.5 h in 5% skim milk and subjected to immunoblotting using primary anti-MAPK antibody overnight at 4 °C. Then, the PVDF membranes were washed three time of 10 min each with TBST and incubated with secondary horseradish peroxidase (HRP)-conjugated antibody for 2 h at room temperature. The blotting of membranes was detected by chemiluminescence (ECL) kits (Millipore, Billerica, MA, USA) and Bio-Rad imaging systems. Relative intensities of protein bands were analyzed by Image J software.
3
Protein Extraction and Western Blot Analysis
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Protein extraction and western blot analysis was performed as described previously (14 (link)). Protein was extracted by radioimmunoprecipitation assay lysis buffer (cat. no. C500005; Sangon Biotech Co., Ltd.) then quantified by bicinchoninic acid method. A total of 50 µg of protein was loaded per lane and separated by 4–12% SDS-PAGE and then transferred to a PVDF membrane. The membrane was blocked by 5% non-fat milk for 1 h at room temperature and subsequently incubated with primary antibodies for iNOS (1:1,000; Abcam, cat. nos. ab3523) and GAPDH (1:1,000; Santa Cruz Biotechnology Inc.; cat. nos. sc-32233) overnight at 4°C. Immunoreactivity was detected by a secondary horseradish peroxidase-conjugated IgG (1:5,000; Thermo Fisher Scientific, Inc.; cat. no. A32731) for 1 h at room temperature. Proteins were developed via enhanced ECL chemiluminescence reagent kit (cat. no. 32109; Thermo Fisher Scientific, Inc.) and visualized using Bio-rad Imaging systems (Bio-Rad Laboratories, Inc.) and quantified with Quantity One image software (v4.62, Bio-Rad Laboratories).
4
Quantitative Western Blot Analysis
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Protein was extracted from transfected cells or xenografts using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China). Protein concentration was assessed using a BCA protein assay kit (KeyGen Biotech Co.). Protein (50 µg) was separated by 10% SDS-PAGE, then transferred onto polyvinylidene fluoride membranes (Millipore Corporation, Bedford, MA, USA). The membrane was washed in 0.1% Tween-20 in Tris-buffered saline solution (TBST), and blocked using 5% non-fat milk. The membrane was incubated with primary antibodies against ITGB8 (#88300) and β-actin (#8457) (both from Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. After washing 3 times, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature in a dilution of 1:2,000. Protein bands were visualized with ECL™ Western Blotting Detection Reagents (Amersham; GE Healthcare, Chalfont, UK) and captured using Bio-Rad Imaging Systems (Bio-Rad).
5
Western Blot Protein Analysis Protocol
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Tumor tissues were cut, and cells were scraped from the dishes. Tissue pieces and cells were lysed in lysis buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5 mM MgCl2, 10% glycerol, 1% Triton X-100, 0.1% SDS] with a protease inhibitor cocktail (Roche Diagnostics, Switzerland) on ice for 20 min. The lysates were centrifuged for 20 min at 20,000 × g. Protein concentration was determined using the BCA method (Thermo Fisher Scientific, Waltham, MA, USA), and lysates were mixed with loading buffer. The protein mixtures were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with primary antibodies and then incubated with HRP-conjugated secondary antibody (Thermo Fisher Scientific). Bands were visualized by chemiluminescence reaction using an ECL detection system (Thermo Fisher Scientific), followed by capture using Bio-Rad Imaging Systems (Bio-Rad, Hercules, CA, USA).
6
Protein Extraction and Western Blot Analysis
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Total proteins were collected from cell lines using RIPA lysis buffer according to the manufacturer’s protocols. Cell lysates were centrifuged at 25,000 × g for 30 min at 4°C, and the protein concentrations in supernatants were measured using a BCA kit (Pierce, Rockford, IL, USA). Samples equal to 20 µg of protein were separated using SDS-PAGE. Then proteins were transferred to NC membranes (Millipore, Billerica, MA, USA) and then blocked with 8% nonfat milk. After that, membranes were incubated with primary SOD2 antibodies overnight at 4°C. After washing for four times, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (Pierce) at 37°C for 1 h. Protein bands were visualized with the ECL and captured using Bio-Rad Imaging Systems (Bio-Rad).
7
Western Blot Analysis of Protein Expression
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Cells were lysed using RIPA lysis buffer (Thermo Scientific), and proteins were extracted. Protein concentration was determined by bicinchoninic acid (BCA) assay (Thermo Scientific). Twenty micrograms of protein was loaded and separated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. After separation, proteins were transferred onto PVDF membrane (Millipore, MA, USA), and then blocked using 8% nonfat milk. Membranes were incubated overnight at 4°C with primary antibodies (β-actin: Santa Cruz, 1:1000; STAT3: CST, 1:1000; Bax: CST, 1:1000; Bcl-2: CST, 1:1000; E-cadherin: CST, 1:1000; Snail: CST, 1:1000). After four-time washing, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Thermo Scientific) at 37°C for 30 min. Protein bands were visualized with the enhanced chemiluminescence and captured using BioRad Imaging Systems (BioRad).
8
Western Blot Protein Detection Protocol
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Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis reagent (Thermo Fisher). The protein concentration was determined using a Bradford protein assay (Sigma-Aldrich). A 50-μg sample was separated on a 10% denaturing polyacrylamide gel (with 5% polyacrylamide stacking gel) and transferred electrophoretically onto a nitrocellulose membrane. After blocking with 5% nonfat dry milk in Tris-buffered saline plus Tween (TBST), the membranes were incubated with primary antibodies and then incubated with HRP-conjugated secondary antibody (Thermo Fisher). Bands were visualized by chemiluminescence reaction using an enhanced chemiluminescence detection system (Thermo Fisher) and captured using Bio-Rad Imaging Systems (Bio-Rad).
9
Western Blot Analysis of Epithelial-Mesenchymal Transition
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We collected the indicated cells using a disposable cell scraper and lysed them in lysis buffer containing PMSF and cocktail inhibitors (radioimmunoprecipitation assay). The total protein was measured with a BCA Protein Quantitation kit. Equivalent amounts of total protein extract were separated by 6%-12% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). Blots were then blocked with 5% skimmed milk for 120 min and membranes were incubated overnight at 4°C with primary antibodies against POU3F2 (ab243045, Abcam), E-cadherin [14472S; Cell Signaling Technologies (CST), Danvers, MA, USA], vimentin (5741T, CST), GSK3β (12456S, CST), β-catenin (8480T, CST), and GAPDH (ab8245, Abcam). On the second day, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (7074S,7076S, CST) at 37°C for 60 min. Protein bands were visualized with enhanced chemiluminescence (ECL kit, Millipore, USA) and analyzed with Bio-Rad Imaging Systems (Bio-Rad).
10
Western Blot Analysis of Protein Expression
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Total protein samples were extracted from tissues or cells using Radio immunoprecipitation (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China) containing phenylmethylsulfonyl fluoride. The total protein concentration was determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Fifty micrograms of protein were subjected to standard sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane, followed by incubation with primary antibodies (Abcam Inc., Cambridge, UK) EZH2 (ab186006), PUMA (ab9643) and GAPDH (ab9485) at 4° C overnight. After incubation with the horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam), the protein signal was developed with Enhanced chemiluminescence (ECL) Immunoblot Detection Kit (Amersham, UK) and analyzed with Bio-Rad imaging systems (Bio-Rad Laboratories, CA, USA). The images were analyzed using Quantity One V4.6.2 and the relative protein quantity was determined by normalizing to GAPDH, as represented by the gray levels.
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