A total of 15×106 Ba/F3 cells SB-transfected or LV-transduced with BCR-ABL1 were lysed using high-salt buffer (20 mM Tris*HCl, 400 mM NaCl, 0.5% NP40, 0.3% Triton X100) with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The protein content was assessed using the Bio-Rad Protein Assay Kit II (Bio-Rad, Hercules, CA, USA). After denaturing the samples in NuPAGE® LDS Sample Buffer (Thermo Fisher Scientific), proteins were separated by electrophoresis using NuPAGE® 4-12% Bis-Tris Protein Gels (Thermo Fisher Scientific), transferred onto PVDF membranes (Thermo Fisher Scientific) with the XCell II™ Blot Module (Thermo Fisher Scientific), and treated with Odyssey Blocking Buffer TBS (LI-COR, Lincoln, NE, USA). Antibodies directed against the following targets were used to probe the membranes: phospho-Akt (Ser473), phospho-Gab2 (Tyr452), phospho-Crkl (Tyr207), Akt, Gab2, Crkl (all from Cell Signaling Technology, Danvers, MA, USA) and GAPDH (Abcam, Cambridge, UK). The DyLight™ conjugated antibodies (Cell Signaling Technology) were used for visualization of specific bands.
Dylight conjugated antibodies
Manufactured by Cell Signaling Technology
DyLight™ conjugated antibodies are a line of fluorescent-labeled secondary antibodies produced by Cell Signaling Technology. These antibodies are designed to be used as detection reagents in various immunodetection techniques, such as Western blotting, immunohistochemistry, and flow cytometry. The DyLight™ dyes provide bright, photostable fluorescent labeling that can be detected using standard fluorescence detection equipment.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay kit 2, by Bio-Rad (2 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (2 mentions)
Odyssey blocking buffer tbs, by LI COR (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Abcam (2 mentions)
Xcell 2 blot module, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Xcell iitm blot module, by Thermo Fisher Scientific (1 mentions)
Immobilon fl pvdf membrane, by Merck Group (1 mentions)
Image studio lite 5, by LI COR (1 mentions)
2 protocols using dylight conjugated antibodies
1
Western Blot Analysis of BCR-ABL1 Signaling
2
Quantifying DNA Damage in Osteosarcoma Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Osteosarcoma cells were lysed using high-salt buffer (20 mM Tris*HCl, 400 mM NaCl, 0.5% NP40, 0.3% Triton X100) with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The protein content was assessed using the Bio-Rad Protein Assay Kit II (Bio-Rad, Hercules, CA, USA). After denaturing the samples in NuPAGE® LDS Sample Buffer (Thermo Fisher Scientific), 25 µg proteins were separated by electrophoresis using NuPAGE® 4–12% Bis-Tris Protein Gels (Thermo Fisher Scientific), transferred onto Immobilon-FL PVDF membranes (Merck) with the XCell IITM Blot Module (Thermo Fisher Scientific), and treated with Odyssey Blocking Buffer TBS (LI-COR). Antibodies directed against the following targets were used to probe the membranes: phospho-histone H2A.X (Ser139) (D7T2V) mouse monoclonal antibodies (Cell Signaling Technology) and GAPDH (Abcam), dilutions 1:1000 and 1:2500 respectively. The DyLight™ conjugated antibodies (Cell Signaling Technology) were used diluted 1:20,000 for visualization of specific bands, which was made on the Odyssey Imaging System (Li-COR). Quantification was performed in Image Studio Lite 5.2 (Li-COR).
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