Cells were washed twice with cold PBS, harvested, and then sonicated in lysis buffer containing 10 mM Tris–HCl buffer (pH 7.5), 1% SDS, 1% Triton X-100, and a protease inhibitor cocktail (Roche, Mannheim, Germany). The skin samples from mice were homogenized and sonicated in the lysis buffer. The protein concentration in each lysate was measured using a BCA protein assay kit (Pierce, IL, USA). Proteins in the supernatant were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred to a PVDF membrane. We detected PECAM1, vascular endothelial cadherin, GAPDH, phospho-VEGFR2, VEGFR2, phospho-Akt, Akt, phospho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-SHP2, SHP2, and ATGL by incubation with the respective antibodies followed by incubation with horseradish peroxidase-conjugated antibodies to rabbit IgG (Amersham Pharmacia Biotech, Uppsala, Sweden).
Horseradish peroxidase conjugated antibodies to rabbit igg
Manufactured by Cytiva
Sourced in Sweden, United States
Horseradish peroxidase-conjugated antibodies to rabbit IgG are a type of laboratory reagent. They are used to detect and quantify the presence of rabbit immunoglobulin G (IgG) in samples. The horseradish peroxidase enzyme conjugated to the antibody serves as a label, allowing for visualization and measurement of the target protein.
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2 protocols using horseradish peroxidase conjugated antibodies to rabbit igg
1
Protein Analysis of Vascular Tissues
2
Western Blot Analysis of Skin Proteins
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We performed a Western blot analysis as previously described (28). The skin samples from mice were homogenized and sonicated in the lysis buffer containing 10 mM Tris-HCl buffer (pH 7.5), 1% SDS, 1%
Triton X-100. The protein concentration in each lysate was measured using a BCA protein assay kit (Pierce, IL, USA). Proteins were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred to a PVDF membrane. We detected α2AP, type I collagen and GAPDH by incubation with anti-α2AP antibodies (Santa Cruz Biotechnology, CA, USA), anti-type I collagen antibodies (Bioss antibodies, MA, USA), anti-α-SMA antibody (Genetex, CA, USA), anti-VE-cadherin antibody (Santa Cruz Biotechnology, CA, USA) and anti-GAPDH antibodies (Sigma-Aldrich, MO, USA) followed by incubation with horseradish peroxidase-conjugated antibodies to rabbit IgG (Amersham Pharmacia Biotech, Uppsala, Sweden).
Triton X-100. The protein concentration in each lysate was measured using a BCA protein assay kit (Pierce, IL, USA). Proteins were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred to a PVDF membrane. We detected α2AP, type I collagen and GAPDH by incubation with anti-α2AP antibodies (Santa Cruz Biotechnology, CA, USA), anti-type I collagen antibodies (Bioss antibodies, MA, USA), anti-α-SMA antibody (Genetex, CA, USA), anti-VE-cadherin antibody (Santa Cruz Biotechnology, CA, USA) and anti-GAPDH antibodies (Sigma-Aldrich, MO, USA) followed by incubation with horseradish peroxidase-conjugated antibodies to rabbit IgG (Amersham Pharmacia Biotech, Uppsala, Sweden).
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