The expression of FVII protein was analyzed by western blotting. Protein samples were linearized by mixing with disulfide-reducing Laemmli buffer (Bio-Rad), boiled at 95°C for 5 minutes, and subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred from the gel to the PVDF membrane (Thermo Fisher Scientific, Waltham, MA). The blot was blocked with 5% skimmed milk in TBS-T (10 mM Tris, 150 mM sodium chloride, 0.05% Tween 20, pH 7.4), probed with the monoclonal rabbit anti-FVII at 1:10 000 dilution (Abcam, Cambridge, United Kingdom) for 3 hours at room temperature (RT), and incubated with peroxidase-conjugated goat anti-rabbit IgG H&L at 1:20 000 dilution (Abcam) for 1 hour at RT. Immunoreactive proteins were detected by using ECL detection reagents (Bio-Rad). The membrane was then exposed to the gel documentation system for chemiluminescence imaging.
Peroxidase conjugated goat anti rabbit igg h l
Manufactured by Abcam
Sourced in United Kingdom
Peroxidase-conjugated goat anti-rabbit IgG H&L is a secondary antibody that binds to the heavy and light chains of rabbit immunoglobulin G (IgG). The antibody is conjugated to the enzyme horseradish peroxidase, which can be used in various detection and labeling techniques.
Automatically generated - may contain errors
2 protocols using peroxidase conjugated goat anti rabbit igg h l
1
FVII Protein Expression by Western Blotting
2
Western Blot Analysis of Factor VII
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The total protein concentration from concentrated cultured supernatant was measured by Bradford protein assay. Concentrated proteins were mixed with disulfide-reducing Laemmli buffer and boiled at 98°C for 5 min. Protein samples were run on a 12% bis-tris SDS-PAGE and transferred to a PVDF membrane (Thermo Fisher Scientific, USA). Membranes were blocked overnight with 5% skimmed milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T). The blocked membranes were washed three times with TBS-T. Next, the membranes were incubated with 1:10,000 dilution of monoclonal rabbit anti-human FVII (Abcam) for 3 h at room temperature. Membranes were washed three times with TBS-T and then incubated with 1:20,000 dilution of peroxidase-conjugated goat anti-rabbit IgG H&L (Abcam). The blot was developed using the ECL Prime Western Blotting System (Amersham, UK) and visualized by X-ray film exposure.
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