Western blot analysis was performed as previously described 12 (link). Briefly, cells were lysed in PRO-PREP protein extract solution to isolate the total cell extract. After the extract was centrifuged at 13,000 rpm for 20 min at 4°C, the protein concentration was determined via the Bradford method. Samples containing 30 µg of protein were subjected to 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The separated proteins were then transferred to polyvinylidene difluoride membranes using the SD Semi-dry Transfer Cell® system (Bio-Rad, Hercules, CA, USA). These membranes were incubated with primary antibodies (anti-endothelial nitric oxide synthase [eNOS] and anti-phospho-eNOS at Ser1177 antibodies; Cell Signaling Technology, Beverly, MA, USA) at a 1:500 dilution (4 μg/mL) in 5% skim milk in Tris-buffered saline with Tween 20 overnight at 4°C, and bound antibody was detected with horseradish peroxidase-conjugated anti-rabbit IgG. The membranes were washed and then developed using the Luminol Reagent system (Animal Genetics, Suwon, Korea).
Sd semi dry transfer cell system
Manufactured by Bio-Rad
Sourced in United States
The SD Semi-dry Transfer Cell® system is a laboratory equipment designed for the transfer of proteins from polyacrylamide gels to membranes in a semi-dry environment. The system provides a simple and efficient method for transferring proteins during Western blotting analysis.
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Lab products found in correlation
3 protocols using sd semi dry transfer cell system
1
Quantification of Phosphorylated eNOS by Western Blot
2
Western Blot Analysis of eNOS Phosphorylation
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Western blot analysis was performed as previously described [21 (link)]. Briefly, cells were lysed in PROPREP protein extract solution to obtain total cell extracts. After centrifugation at 13,000 rpm for 20 min at 4°C, the protein concentration was determined by the Bradford method. Thirty micrograms of protein was subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The separated proteins were transferred to a polyvinylidene difluoride membrane using the SD Semi-Dry Transfer Cell System (Bio-Rad, Hercules, CA, USA). The membranes were incubated with primary antibodies (anti-eNOS and anti-phospho-eNOS antibodies; Cell Signaling Technology, Beverly, MA, USA; 4 μg/mL) in 5% skim milk in TBST overnight at 4°C, and the bound antibody was detected with horseradish peroxidase-conjugated anti-rabbit IgG. The membranes were washed and then developed using the Luminol Reagent system (Animal Genetics, Suwon, Korea).
3
Western Blot Analysis of eNOS and p-eNOS
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Western blot analysis was performed as previously described 18 (link). Briefly, cells were lysed in PRO-PREP protein extract solution to isolate total cell extracts. After extracts were centrifuged at 13,000 rpm for 20 min at 4°C, protein concentrations were determined by the Bradford method. Samples containing 30 µg of protein were subjected to 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The separated proteins were then transferred to polyvinylidene difluoride membranes using the SD Semi-dry Transfer Cell® system (Bio-Rad, Hercules, CA, USA). These membranes were incubated with primary antibodies (anti-endothelial nitric oxide synthase [eNOS] and anti-phospho-eNOS [at Ser1177] antibodies; Cell Signaling Technology, Beverly, MA, USA) at a 1:500 dilution (4 μg/mL) in 5% skim milk in TBST overnight at 4°C, and bound antibody was detected by horseradish peroxidase-conjugated anti-rabbit IgG. The membranes were washed and then developed using the Luminol Reagent system (Animal Genetics, Suwon, Korea). Beta-actin was used as the loading control.
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