The involved protein expression was examined by Western blot analysis. Total protein was extracted from the transfected A549 and H1299 cells, as well as the mice tumor tissues by RIPA lysis buffer (Beyotime, Shanghai, China). 30 μg total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, the proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, the membrane was incubated with 5% skim milk (dissolved in TBST buffer) for 1 h at room temperature. The proteins were incubated with the primary antibodies against c-myc (1:1000, 18,583, Cell Signaling Technology (CST), Boston, MA, USA), Cyclin D1 (1:200, ab16663, Abcam, Cambridge, UK), matrix metalloprotein 9 (MMP9, 1:1000, 13,667, CST), E-cadherin (E-cad, 1:1000; 14,472, CST), HK2 (1:1000, ab104836, Abcam), LDHA (1:2000, ab101562, Abcam) and GAPDH (1:1000, ab8245, Abcam) overnight at 4°C, followed by incubating with the secondary antibodies including goat anti-mouse IgG H&L (1:2000, ab205719, Abcam) or goat anti-rabbit IgG H&L (1:2000, ab6721, Abcam) for 2 h. Finally, the signals of proteins were visualized by an electrochemiluminescent system (PerkinElmer Life Science, Waltham, MA, USA).
Electrochemiluminescent system
Manufactured by PerkinElmer
Sourced in United States
The Electrochemiluminescent system is a lab equipment designed for the detection and quantification of a wide range of analytes, including proteins, nucleic acids, and small molecules. This system utilizes electrochemically-induced luminescence to generate a signal that is proportional to the concentration of the target analyte in the sample. The core function of this system is to provide a sensitive and accurate method for analyzing a variety of samples in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor cocktail, by Roche (4 mentions)
Dc protein assay, by Bio-Rad (4 mentions)
Chemiluminescent hrp antibody detection kit, by Thomas Scientific (4 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Ab6721, by Abcam (3 mentions)
Ab104836, by Abcam (3 mentions)
Ab205719, by Abcam (3 mentions)
Ab8245, by Abcam (3 mentions)
Ab101562, by Abcam (2 mentions)
Sds page, by Fujifilm (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Ab16663, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Anti β actin antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Radioimmunoprecipitation lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Beyoecl plus chemiluminescence kit, by Beyotime (1 mentions)
Ab40084, by Abcam (1 mentions)
13 protocols using electrochemiluminescent system
1
Protein Expression Analysis in Lung Cancer
2
Western Blot Analysis of LRIG2, HK2 in EC Tissues
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Total protein was isolated from the EC tissues and normal tissues, as well as RL95-2 and Ishikawa cells by RIPA lysis buffer (Beyotime, Shanghai, China). Briefly, the total protein was separated using 8–12% odium dodecyl sulfate–polyacrylamide gel electrophoresis, and then the proteins were transferred onto the polyvinylidene difluoride membranes (Sigma, Billerica, MA, USA). Subsequently, the membranes were blocked using 5% non-fat milk and incubated with the primary antibodies against LRIG2 (1:1000, ab157492, Abcam, Cambridge, UK), HK2 (1:1000, ab104836, Abcam) and GAPDH (1:1000, ab8245, Abcam) overnight at 4 °C. Then, the blots were incubated with goat anti-rabbit (1:2000, ab6721, Abcam) or anti-mouse (1:2000, ab205719, Abcam) for 2 h. Finally, the protein signals were calculated by an electrochemiluminescent system (PerkinElmer Life Science, Waltham, MA, USA).
3
Western Blot Detection of PKM2 Protein
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Cells were lysed in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS). Complete protease inhibitor cocktail (Roche, Indianapolis, IN, USA) was added to lysis buffer before use. Protein concentration was determined by Bio-Rad DC protein assay (Bio-Rad, Hercules, CA, USA). 20 ug of total protein from cell lysate was subjected to SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked in 5% non-fat milk in PBS overnight and incubated with primary antibody. After washing for 30 min, secondary goat anti-mouse IgG (Vector Co., Burlingame, CA, USA) was applied to nitrocellulose membrane in TBS-Tween for 1 h. After washing, the proteins of interest were detected using Chemiluminescent HRP Antibody Detection Kit (Denville Scientific, South Plainfield, NJ, USA). Anti-PKM2 polyclonal antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-β-actin antibodies were purchased from Cell signaling technology (Danvers, MA, USA). The protein signals were captured using an electrochemiluminescent system (PerkinElmer Life Science, Boston, MA, USA).
4
Western Blot Protein Analysis Protocol
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Cells were lysed with 1× RIPA Buffer. Lysates (25 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Wako Pure Chemical Industries, Ltd., Osaka, Japan) and transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Little Chalfont, UK). The membranes were incubated with the primary antibodies and then with a corresponding secondary horseradish peroxidase (HRP)-conjugated IgG. Proteins were visualized with an electrochemiluminescent system (PerkinElmer, Waltham, MA, USA).
5
Exosomal Protein Analysis via Western Blot
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The exosomal samples were plated onto six-well plates and lysed with radioimmunoprecipitation assay buffer (RIPA buffer; 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% sodium deoxycholate, 1% NP-40, and 0.1% sodium dodecyl sulfate). Lysates were separated by 5–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes, followed by incubation with primary antibodies (CD63) and then incubation with the corresponding secondary horseradish peroxidase-conjugated IgG. The proteins were visualized with an electrochemiluminescent system (PerkinElmer Life Science, Waltham, MA, USA).
6
Quantification of Extracellular Matrix Proteins
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A total of 5 × 105 cells were plated onto 6-well plates and lysed with radioimmunoprecipitation assay buffer (RIPA buffer; 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% sodium deoxycholate, 1% NP-40, and 0.1% sodium dodecyl sulfate). Lysates were separated by 5–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes, followed by incubation with primary antibodies (vitronectin, 1:1000 in 5% BSA; fibronectin, 1:1000 in 5% BSA; β-actin, 1:2000 in 5% BSA), and then incubation with the corresponding secondary horseradish peroxidase–conjugated IgG. The proteins were visualized with an electrochemiluminescent system (PerkinElmer Life Science, Waltham, MA).
7
Exosome Protein Profiling Using Western Blot
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A2780 cells and plasma-derived exosomes were lysed with radioimmunoprecipitation lysis buffer (Beyotime, Jiangsu, China), and total protein was obtained by centrifugation at 12,000×g for 15 min at 4 °C. After quantification using a bicinchoninic acid (BCA) Protein Assay Kit (Beyotime), equal amounts of cellular and exosomal protein were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membrane was blocked with 10% defatted milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h and then incubated overnight at 4 °C with primary antibodies, including anti-GM130 (Abcam ab52649; 1:1000), anti-HSP70 (Abcam ab2787; 1:1000), and anti-CD9 (Abcam ab236630; 1:1000). After washing with TBST three times, the membrane was incubated with appropriate HRP-conjugated secondary antibodies: goat anti-rabbit IgG H&L (HRP) (Abcam ab97051; 1:2000) or goat anti-mouse IgG H&L (HRP) (Abcam ab205719; 1:2000), as appropriate, for 1 h at room temperature. Finally, the protein bands were visualized on photographic films using the BeyoECL Plus chemiluminescence kit (Beyotime) with an electro-chemiluminescent system (PerkinElmer Life Science, Waltham, MA, USA).
8
Protein Expression Analysis of Metabolic Markers
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Total protein was isolated using RIPA lysis buffer (Beyotime, Shanghai, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate the proteins. Then, the proteins were incubated with the primary antibodies against GLUT1 (1:5000, ab40084, Abcam, Cambridge, UK), HK2 (1:1000, ab104836, Abcam), LDHA (1:2000, ab101562, Abcam), CD81 (1:1000, 10,037, Cell Signaling Technology, Boston, MA, USA), TSG101 (1:1000, ab30871, Abcam), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH,1:1000, ab8245, Abcam) overnight at 4°C. Then, the proteins were incubated with the secondary antibodies against goat anti-rabbit IgG H&L (1:2000, ab6721, Abcam) or goat anti-mouse IgG H&L (1:2000, ab205719, Abcam). Finally, the signals were visualized using an electrochemiluminescent system (PerkinElmer Life Science, Waltham, MA, USA).
9
Quantitative Western Blot Analysis
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A total of 5 × 105 cells were plated onto 6-well plates and lysed with 1 × RIPA Buffer (25 mM Tris-Hcl pH 7.6, 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% sodium deoxycholate, 1 mM PMSF, protease inhibitor cocktail; 1:100 [Nacalai Tesque, Inc., Kyoto, Japan]). Lysates (15 μg) were separated by 10% or 5–20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Wako Pure Chemical Industries, Ltd., Osaka, Japan), and transferred to polyvinylidene difluoride membranes, followed by incubation with the primary antibodies (PAI-1, 1:200; uPAR 1:1000; PARP, 1:1,000; CDK2, 1:500; Cyclin D3, 1:2,000; p27 kip1, 1:500; p-ERK 1/2, 1:1,000; ERK1/2, 1:1,000; p-FAK, 1:1,000; FAK 1:1,000; Integrin αV, 1:250; Integrin β3, 1:2500; β-actin, 1:2,000) in TBS-T containing 20% Bullet Blocking One (Nacalai Tesque) and then with a corresponding secondary horseradish peroxidase-conjugated IgG. The proteins were visualized with an electrochemiluminescent system (PerkinElmer, Waltham, MA, USA).
10
Western Blot Analysis of SEC23A, YAP, and β-Tubulin
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Cells were lysed in RIPA buffer (1 % NP-40, 0.5 % sodium deoxycholate, 0.1 % SDS in PBS). Complete protease inhibitor cocktail (Roche, Indianapolis, IN, USA) was added to lysis buffer before use. Protein concentration was determined by Bio-Rad DC protein assay (Bio-Rad, Hercules, CA, USA). 20ug of total protein from cell lysate was subjected to SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked in 5 % non-fat milk in PBS overnight and incubated with primary antibody. After washing for 30 min, secondary goat anti-mouse IgG (Vector Co., Burlingame, CA, USA) was applied to nitrocellulose membrane in TBS-Tween for 1 h. After washing, the proteins of interest were detected using Chemiluminescent HRP Antibody Detection Kit (Denville Scientific, South Plainfield, NJ, USA). Anti-SEC23A polyclonal antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-β-tubulin and anti-YAP antibodies were purchased from Cell signaling technology (Danvers, MA, USA). The protein signals were captured using an electrochemiluminescent system (PerkinElmer Life Science, Boston, MA, USA).
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