Western blot analysis was carried out on extracted cell-culture and tumor samples,19 (link) and membranes were probed with primary antibodies against mouse HIF1α (1/800, AF1935), CAIX (1/800, AF2344), and β-actin (1/10,000) as loading control; all antibodies were from R&D Systems (Minneapolis, MN, USA). Secondary horseradish peroxidase-conjugated antibodies were from Agilent Technologies (Santa Clara, CA, USA). Protein bands were quantified using Alliance 2.7 software (Uvitec, Cambridge, UK) and the protein of interest normalized against a positive control (hypoxia-treated LL/2 whole-cell lysate) following confirmation of equal loading using β-actin.
Protocol detail
Quantitative Western Blot Analysis
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Actin
Antibodies
Caix
Cell
Cell culture
Hif1α
Horseradish peroxidase
Hypoxia
Membranes
Mouse
Protein
Tumor
Western blot analysis
Corresponding Organization :
Other organizations : University of Otago
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Variable analysis
independent variables
- Mouse HIF1α antibody concentration (1/800)
- CAIX antibody concentration (1/800)
- β-actin antibody concentration (1/10,000)
- Protein band quantification of mouse HIF1α
- Protein band quantification of CAIX
- Protein band quantification of β-actin
- Hypoxia-treated LL/2 whole-cell lysate (used as a positive control)
- Positive control: Hypoxia-treated LL/2 whole-cell lysate
- Negative control: Not specified
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