Changes of protein levels were examined by Western blotting, as described in our previous study (Ye & Steinle, 2015 (link)). After transfer, the membrane was incubated with appropriate primary antibodies followed by treatment with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were detected by chemilluminescence reagent kit (Thermo Scientific, Pittsburgh, PA). Primary antibodies used were TGF beta3, phosphorylated SMAD2/3, and SMAD2/3 (rabbit monoclonal, 1:500; all purchased from Cell Signaling, Danvers, MA), VEGF, ZO-1, and occludin (all from Abcam, Cambridge, MA), and beta actin (Santa Cruz Biotechnology, Santa Cruz, CA). Imaging was performed using C500 system (Azure Biosystems, Dublin, CA) within a linear range of exposure. beta actin was used to normalize signal intensity of protein bands.
C500 system
Manufactured by Azure Biosystems
The C500 system is a capillary electrophoresis instrument designed for the separation and analysis of biomolecules, such as nucleic acids and proteins. It features an automated sample handling system, a sensitive detection method, and proprietary software for data analysis. The core function of the C500 system is to provide accurate and reliable results for various applications in life science research and diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
Chemilluminescence reagent kit, by Thermo Fisher Scientific (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
Phosphorylated smad2 3, by Cell Signaling Technology (1 mentions)
Vascular endothelial growth factor (vegf), by Abcam (1 mentions)
Occludin, by Abcam (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
2 protocols using c500 system
1
Quantifying Protein Expression Changes
2
Western Blot Analysis of NF-κB
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After rinsing with cold PBS, REC or whole retinal lysates were collected in lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein were separated on precast Tris-glycine gels (Invitrogen, Carlsbad, CA) and then blotted onto a nitrocellulose membrane. After blocking in TBST (10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, the membrane was treated with appropriate primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were detected by chemilluminescence reagent kit (Thermo Scientific, Pittsburgh, PA). Primary antibodies used were phosphorylated NF-κB p65 (Ser 536) (Cat.# 3033), NF-κB p65 (Cat.# 4764) (rabbit monoclonal, 1:500; all purchased from Cell Signaling, Danvers, MA), and beta actin (Santa Cruz, Santa Cruz, CA). Imaging was performed using C500 system (Azure Biosystems, Dublin, CA) within a linear range of exposure. Signal intensity of protein bands was normalized by beta actin. Even staining of beta actin was observed after loading of equal amounts of protein.
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