Cells were lysed in RIPA buffer, containing 50 mM Tris·HCl, 0.5% DOC, 0.1% SDS, 1 mM EGTA, 2 mM EDTA, 10% glycerol, 1% Triton X-100, 150 mM NaCl and 1 mM PMSF, and protease inhibitors (ROCHE pill Complete Inhibitor). Protein concentrations were determined with the Bradford Protein Assay (Bio-Rad). Proteins (30 g) were separated by 6% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat dry milk (Marvel) and subsequently incubated (overnight, 4°C) with primary antibodies anti-HIF-1α (1:1000; #610959, BD Biosciences) and anti-lamin A (1:1000; #L1293, Sigma-Aldrich), and then horseradish peroxidase-linked secondary antibodies (horse anti-mouse and horse anti-rabbit; 1:2500; Cell Signaling Technology). ECL Prime Western Blotting Detection Reagent (GE Healthcare) was used for visualisation.
Horseradish peroxidase linked secondary antibodies horse anti mouse and horse anti rabbit
Manufactured by Cell Signaling Technology
Horseradish peroxidase-linked secondary antibodies (horse anti-mouse and horse anti-rabbit) are lab equipment used for detection and visualization in various immunoassays. These antibodies are conjugated with the enzyme horseradish peroxidase, which catalyzes a colorimetric reaction when exposed to a suitable substrate, allowing for the indirect detection of target proteins.
Automatically generated - may contain errors
2 protocols using horseradish peroxidase linked secondary antibodies horse anti mouse and horse anti rabbit
1
Immunoblot Analysis of HIF-1α and Lamin A
2
Protein Extraction and Western Blot Analysis
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Cells were lysed in RIPA buffer, containing 50 mM Tris•HCl, 0.5% DOC, 0.1% SDS, 1 mM EGTA, 2 mM EDTA, 10% glycerol, 1% Triton X100, 150 mM NaCl, and 1 mM PMSF, and protease inhibitors (ROCHE pill Complete Inhibitor). Protein concentrations were determined with Bradford Protein Assay (Bio-Rad). Proteins (30 g) were separated on a 6% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat dry milk (Marvel) and subsequently incubated (O/N, 4°C) with primary antibodies HIF-1α (BD Biosciences) Lamin A (Sigma) and then horseradish peroxidase-linked secondary antibodies (horse anti-mouse and horse anti-rabbit; 1:2,500;
Cell Signaling). ECL Prime Western Blotting Detection Reagent (GE Healthcare) was used for visualisation.
Cell Signaling). ECL Prime Western Blotting Detection Reagent (GE Healthcare) was used for visualisation.
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