Proteins from aorta homogenates were separated on polyacrylamide-sodium dodecyl sulfate gels and transferred onto polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies at 4 °C in 10% milk or 3% bovine serum albumin in Tris-buffered saline containing 0.05% Tween-20 overnight. The primary antibodies used in this study were anti-mouse eNOS (1:1000; BD transduction Laboratories, Lexington, KY, USA), anti-mouse eNOS-P Ser1177 (1:1,000; BD Transduction Laboratory), anti-rabbit GAPDH (glyceraldehyde 3-phosphate dehydrogenase; 1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-rabbit nitrotyrosine (1:15 000; Millipore Corporation, Billerica, MA, USA). Immunodetection was carried out using an ECL or ECL Plus system (SuperSignal West Pico Chemiluminescence Substrate, Thermo Scientific (Villebon-sur-Yvette, France) or Luminata Forte Western HRP substrate, Millipore Corporation, respectively), and the membranes were then exposed to X-ray films for visualization. The eNOS protein content was expressed relative to the GAPDH content. The eNOS-P Ser1177 protein content was expressed relative to the eNOS content.
Anti mouse enos p ser1177
Manufactured by BD
Sourced in United States
The Anti-mouse eNOS-P Ser1177 is a laboratory equipment product that detects the phosphorylation of endothelial nitric oxide synthase (eNOS) at serine 1177. It is a specific antibody used for the detection and quantification of this post-translational modification in mouse samples.
Automatically generated - may contain errors
2 protocols using anti mouse enos p ser1177
1
Western Blot Analysis of Aortic Proteins
2
Western Blot Analysis of eNOS Phosphorylation
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Equal amounts of protein (30 μg/lane) estimated by Bradford assay were solubilized in a 2X Laemmli sample buffer containing 2-mercaptoethanol and loaded in a denaturing SDS/10% polyacrylamide gel. Western blot analysis was performed with monoclonal anti-mouse eNOS-PSer1177 (1 : 1000; BD Transduction Laboratory) (2 μg/mL) and anti-mouse eNOS (1 : 1000; BD Transduction Laboratory) (2 μg/mL) incubated overnight at 4°C. After incubation, the pure nitrocellulose membranes (0.45 μm) were washed and incubated with goat anti-mouse IgG1-horseradish peroxidase for 2 hours. Immune complexes were detected by enhanced chemiluminescence. The developed spots were quantified by densitometric analysis on a NIH Image 1.62f analyzer, and the value was expressed as arbitrary units. Each sample was analyzed in triplicate. The results were expressed as P-eNOS/total eNOS ratio.
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