General considerations for Western blot analysis of HIF-1 alpha proteins:
1. HIF-1alpha is largely undetectable in cells or tissues grown under normoxic conditions. It is stabilized only at O2 concentrations below 5% or with treatment using certain agents (CoCl2, DFO, etc.), therefore proper sample preparation is critical. We recommend lysing cells quickly and directly into the Laemmli sample buffer with DTT or BME.
2. Since stabilized HIF-1alpha translocates to the nucleus, using nuclear extracts is recommended for western blot analysis.
3. Positive and negative controls should always be run side by side in a Western blot to accurately identify the protein band upregulated in the hypoxic sample. (HeLa Hypoxic/Normoxic Cell Lysate: NBP2-36452; HeLa Hypoxic (CoCl2)/Normoxic Lysate: NBP2-36450)
4. To accurately compare treated and untreated samples and to ensure equal loading of samples the expression of a loading control should be evaluated. (alpha Tubulin Antibody (DM1A): NB100-690)
5. Unprocessed HIF-1alpha is ~95 kDa, while the fully post-translationally modified form is ~116 kDa, or larger.
6. HIF-1alpha may form a heterodimer with HIF-1beta (Duan, et al. Circulation. 2005; 111:2227-2232.). However, this is not typically seen under denaturing conditions.
7. Depending on the sample and treatment, a single band or a doublet may be present.
Western Blot Protocol
1. Load samples of treated and untreated cell lysates, 10-40 mg of total protein per lane on a 7.5%polyacrylamide
gel (SDS-PAGE). Alternatively, gradient gels can be used for better resolution of lower molecular weight loading
controls.
2. Resolve proteins by electrophoresis as required.
3. Transfer proteins to 0.45 mm PVDF membrane for 1 hour at 100V or equivalent.
4. Stain the blot using Ponceau S for 1-2 minutes to confirm efficient protein transfer onto the membrane.
5. Rinse the blot in distilled water to remove excess stain and mark the lanes and locations of molecular weight
markers using a pencil.
6. Block the membrane using 5% non-fat dry milk in TBST (0.1% Tween) for 1 hour.
7. Dilute the mouse anti-HIF-1 alpha primary antibody (NB100-105) at 2ug/ml in blocking solution and incubate 1
hour at room temperature or overnight at 4C.
8. Wash the membrane 3X 5 min in TBST.
9. Incubate in the appropriate diluted mouse-IgG HRP-conjugated secondary antibody in blocking solution (as per
manufacturer's instructions) for 1 hour at room temperature.
10. Wash the membrane 3X 5 min in TBST.
11. Incubate with ECL detection reagent (Supersignal West Pico Plus, or more sensitive) for 5 min.
12. Image the blot. That may require up to 5min of exposure due to weak signal.
