Cells were lysed with Nonidet P (NP)-40 buffer (1% NP-40, 0.15 M NaCl, 50 mM Tris; pH 8.0) containing protease inhibitors (Sigma-Aldrich; Merck KGaA). A bicinchoninic acid assay was used for protein quantitation (Pierce; Thermo Fisher Scientific, Inc.). Samples were denatured in SDS buffer and 50 µg protein was loaded per lane and separated by electrophoresis on SDS gels with 8–10% polyacrylamide. The proteins were transferred onto polyvinylidene difluoride membranes. These were then incubated with the following antibodies: Anti-PAPSS2 (cat. no. ab37611; 1:100) anti-COL2 (cat. no. ab185430; 2 µg/ml) and anti-COLX (cat. no. ab58632; 2 µg/ml), and anti-β-actin (cat. no. AC-40; 1:2,000; all Abcam, Cambridge, MA, USA) for 12 h at 4°C. Subsequently, membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary anti-mouse [m-IgGκ binding protein horseradish peroxidase (HRP) conjugated; cat. no. sc-516102] or anti-rabbit (mouse anti-rabbit HRP-IgG; cat. no. sc-2357) antibodies (each 1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 2 h. Proteins were visualized using enhanced chemiluminescence (Advansta, Inc., Menlo Park, CA, USA) and the blots were imaged and quantified with the Fluor-S Multi-Imager system and Multi-Analyst software version 1.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Enhanced chemiluminescence
Manufactured by Advansta
Sourced in United States
Enhanced chemiluminescence is a laboratory technique used to detect and quantify proteins in Western blotting applications. It involves the use of a chemiluminescent substrate that emits light upon reaction with the target protein, which can then be detected and measured using specialized imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Polyvinylidene difluoride membrane, by Merck Group (4 mentions)
Pro measure, by iNtRON Biotechnology (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Pro prep, by iNtRON Biotechnology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Dc protein assay kit, by Bio-Rad (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Multi analyst software version 1, by Bio-Rad (1 mentions)
Fluor s multi imager system, by Bio-Rad (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Ripa cell lysate, by Applygen (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Superblock, by Thermo Fisher Scientific (1 mentions)
C300 imaging system, by Azure Biosystems (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
33 protocols using enhanced chemiluminescence
1
Western Blot Analysis of Cartilage Proteins
2
Protein Extraction and Western Blot Analysis
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After 48 h of EEC treatment, the original culture medium was discarded and cells were washed with PBS three times, placed on ice, and mixed with RIPA cell lysate (Applygen Technologies Inc., Beijing, China), containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). The protein extraction method and WB analysis have been described previously [9 (link),23 (link)]. All test antibodies are shown in Table 2 . The blots were visualized using enhanced chemiluminescence (Advansta, Menlo Park, CA, USA), and related data were analyzed using ImageJ processing software; each experiment was performed in triplicate.
3
Western Blot Analysis Protocol
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The cells were washed three times with PBS at 4°C, and the entire population was extracted and lysed with lysis buffer for 30 min on ice. Next, the lysates were centrifuged at 14000 g for 15 min at 4°C, and the precipitates and supernatants were collected. Then, the total protein (20–50 μg) was separated by SDS-PAGE, and the separated proteins were transferred by the wet method to PVDF membranes. Subsequently, the membranes were blocked with Superblock (Thermo Fisher Scientific, USA) for 2 h at room temperature. Next, the membranes were incubated with primary Abs overnight at 4°C and then with the appropriate secondary Abs for 2 h at room temperature. Finally, the proteins were visualized by an enhanced chemiluminescence (Advansta Inc., San Jose, CA, USA) system. The results were analysed by ImageJ software, and β-actin was used as the internal control.
4
Protein Quantification and Western Blotting
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After terminating platelet aggregation, lysis buffer (PRO-PREP; iNtRON Biotechnology, Seoul, Korea) was added to the mixture. A BCS assay (PRO-MEASURE; iNtRON Biotechnology) was used to measure protein concentration. Total platelet protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. PVDF membranes were then incubated with primary and secondary antibodies conjugated to horseradish peroxidase, and antibody binding was visualized using enhanced chemiluminescence (Advansta Inc., Menlo Park, CA, USA).
5
Protein Extraction and Immunoblotting from C.elegans, C2C12 Cells, and Mouse Tissues
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C.elegans were lysed by sonication with TBS buffer containing protease and phosphatase inhibitors (Roche), and analyzed by western and dot blot. The concentration of extracted protein was determined using the Bio-Rad Protein Assay. Membranes were blocked in 10% milk for 2h or overnight. Homogeneous loading was monitored by ponceau red. Antibody detection reactions for all immunoblot experiments were developed by enhanced chemiluminescence (Advansta) and imaged using the c300 imaging system (Azure Biosystems). Each immunoblot experiment was repeated at least twice using at least three biological replicates each containing approximately 1,000 worms.
C2C12 cell lysates were prepared for immunoblotting as for C.elegans. Each experiment was repeated at least twice using 3 biological replicates.
For mouse tissues, frozen forelimbs samples were lysed by mechanical homogenization with RIPA buffer containing protease and phosphatase inhibitors for western blot analysis. Lysates were prepared and analyzed by dot blot as described for worms.
C2C12 cell lysates were prepared for immunoblotting as for C.elegans. Each experiment was repeated at least twice using 3 biological replicates.
For mouse tissues, frozen forelimbs samples were lysed by mechanical homogenization with RIPA buffer containing protease and phosphatase inhibitors for western blot analysis. Lysates were prepared and analyzed by dot blot as described for worms.
6
Western Blot Analysis of Chalepin-Treated Cells
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For this analysis, 1 × 106 of A549 cells were plated per tissue culture dish. Chalepin-treated whole-cell extracts were lysed in lysis buffer (20 mM Tris (pH 7.4), 250 mM natrium chloride, 2 mM ethylenediaminetetraacetic acid (pH 8.0), 0.1% Triton X-100, 0.01 μg/ml aprotinin, 0.005 μg/ml leupeptin, 0.4 mM phenylmethylsulfonyl fluoride, and 4 mM sodium orthovanadate). Lysates were then spun at 15,000 rpm for 10 min to remove insoluble material and resolved on a 10.0% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. After electrophoresis, the proteins were electrotransferred to a nitrocellulose membrane, blocked with Blocking One (Nacalai Tesque, Inc.), and probed with various antibodies (1:1000) overnight at 4°C. The blot was washed, exposed to horseradish peroxidase-conjugated secondary antibodies for 1 h, and finally examined by chemiluminescence (enhanced chemiluminescence, Advansta).
7
Western Blot Analysis of Cardioprotective Proteins
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The related proteins, that is Nrf2, HO‐1, SOD1, SOD2, Bcl2, Bax, caspase‐3, AMPK, pAMPK, Sirt1, β‐actin or GAPDH, were determined in rat cardiomyoblasts or mouse ventricular tissues by Western blot analysis as described previously.14 The samples were lysed by RIPA buffer with protease inhibitors, and the protein concentrations were determined by BCA assay. Equal amounts of total proteins were separated by using SDS‐PAGE and transferred onto PVDF membranes (Bio‐Rad, Hercules, CA, USA) which were blocked by 5% skim milk and incubated with primary antibodies (1:1000) at 4°C overnight. Then, membranes were washed three times with TBST and incubated with secondary antibody (1:10 000) at room temperature for 1 hour. The membranes were visualized by enhanced chemiluminescence (Advansta, Menlo Park, CA, USA) and were exposed to FluoChem E chemiluminescence detection system (ProteinSimple, San Jose, CA, USA). The relative densities of protein bands were analysed by image analysis software. The Western blots used in the results figures are included in Supplemental original data.
8
Western Blot Analysis of Protein Signaling
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PBS washed cells were harvested and lysed in RIPA buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, and 1% sodium deoxycholate) containing the Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher, Waltham, MA, USA). Protein concentration was determined using the T-Pro BCA Protein Assay Kit (T-Pro biotechnology, Taipei, Taiwan). Protein samples were separated by SDS-PAGE, transferred onto PVDF membranes (Millipore, Billerica, MA, USA), and blocked with Block PRO blocking buffer (Visual Protein Biotechnology Corporation, Taipei, Taiwan). Blot was incubated with a primary antibody followed by incubation of horseradish peroxidase-conjugated secondary antibodies. Protein level was determined by enhanced chemiluminescence (Advansta, Menlo Park, CA, USA) detection. Antibodies probing p-AKT (S473), AKT, p-ERK1/2 (Y202/Y204), ERK1/2, and GAPDH (Cell Signaling Technology, Inc., Danvers, MA, USA) were used.
9
Protein Expression Analysis by SDS-PAGE and Western Blot
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The proteins were separated using 10% SDS-polyacrylamide gel electrophoresis at 200 V for 1.5 h, and then 110 mA for 3 h at 4°C. Protein bands from the gel were transferred onto polyvinylidene fluoride (PVDF) membranes using transfer buffer. The PVDF was blocked with 5% fat-free powdered milk in a blocking solution, including tris-buffered and tween 20 (TBST), at room temperature for 2 h. After washing three times with TBST, the PVDF was incubated overnight at 4°C with primary antibody (Cell Signaling Technology, USA). After incubation, PVDF was washed with TBST and incubated with the secondary antibody (TransGen Biotech, China) at RT for 2 h. The proteins of PVDF were detected using enhanced chemiluminescence (Advansta, Menlo Park, CA, USA). Grey scale analysis was performed using ImageJ, and statistical data analyses were performed by Prism 9 software.
10
Protein Expression Analysis in Mouse Tissues
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Mouse tissues were crushed into a fine powder in liquid nitrogen and lysed in RIPA buffer supplemented with complete protease inhibitor (Roche, Switzerland). Protein concentration was determined using the BCA method (KeyGEN Bio TECH Nanjing, China). Western blot analysis was performed as previously described (23 (link)). Aliquots (20 μg) resolved by 8% SDS-PAGE gels electrophoresis, transferred to PVDF membrane (Pall, USA). After blocking with 5% non-fat milk in phosphate buffered saline/Tween-20, the membrane was incubated with antibodies specific to SHP-2 (1:1,000; #3397, Cell Signaling Technology, Boston, USA), CDC25A (1:1,000; #137353, Abcam, UK), mTOR (1:1,000; #2983, Cell Signaling Technology, Boston, USA), IGF1R-β (1:1,000; #9750, Cell Signaling Technology, Boston, USA). The antigen-antibody complexes were detected using an enhanced chemiluminescence (Advansta, Menlo Park, USA). The abundance assessed quantitatively using the Image J softwar.
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