Cultured BM-MPCs were placed in hyperoxia or hypoxia for 24 h. Nuclear protein was obtained using Pierce Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL) according to the manufacturer’s instructions. Protein content was standardized using the BCA Protein Assay kit (Pierce Biotechnology). Nuclear protein (20 μg) was separated by SDS-PAGE on 7.5% gels, transferred to polyvinylidene difluoride membranes, and blocked with 5% milk in tris-buffered saline with Tween. Protein detection was performed with primary antibodies for HIF-1α (Novus Biologicals, Littleton, CO), with β-actin serving as an internal loading control (Lab Vision, Fremont, CA). Blots were subsequently incubated with a horseradish peroxidase–conjugated secondary antibody (Amersham Biosciences, Piscataway, NJ). Blots were developed with ECL reagent (Amersham Biosciences) and exposed for 1–10 min on Kodak Biomax-MS film. Band densities were quantified with ImageJ software (National Institutes of Health).
Primary antibodies for hif 1α
Manufactured by Novus Biologicals
Primary antibodies for HIF-1α are laboratory reagents used to detect and quantify the presence of the Hypoxia-Inducible Factor-1 alpha (HIF-1α) protein in biological samples. HIF-1α is a critical transcription factor that regulates the cellular response to hypoxic conditions.
Automatically generated - may contain errors
Lab products found in correlation
Ecl reagent, by Cytiva (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cytiva (1 mentions)
Biomax ms film, by Kodak (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
β actin, by Lab Vision (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
2 protocols using primary antibodies for hif 1α
1
Hypoxia Regulates Nuclear HIF-1α
2
Protein Isolation and Western Blot Analysis
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Protein was isolated from each cell type at the indicated time points from 6‐well plates in 1× cell lysis buffer (Cell Signaling) containing the protease inhibitor AEBSF (Sigma). Lysate concentrations were quantified using the Bradford Coomassie colormetric assay (Pierce), and supernatants were run on SDS‐page gels (Bio‐Rad) and transferred to polyvinylidene fluoride membranes (Bio‐Rad). Blots were probed using primary antibodies for HIF1α (1:500; Novus Biologicals) and normalized to horseradish peroxidase tagged β‐actin (1:15,000; Sigma).
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