HIF-1 alpha Western Blot Info

1. The HIF proteins are among the most rapidly degrading proteins ever studied. Upon cellular re-oxygenation it can be completely degraded in less than 1 minute. Therefore, it is critical to prep only a few plates/dishes/flasks of cells at a time and to immediately place the cells into ice cold buffers and perform the whole protein prep on ice.
2. HIF-1 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.) so proper sample preparation is critical.
3. Upon stabilization HIF-1 translocates to the nucleus. The best western blots (cleanest) are always done using nuclear extracts. It is possible to detect HIF-1 in whole cell extracts, but they tend to be much dirtier and the staining is much weaker.
4. Finally, we recommend that a positive/negative control always be run side by side so that it is possible to discern which band is upregulated in the hypoxic sample. Unprocessed HIF1 is ~95 kDa while the fully post-translationally modified form is ~116 kDa, or larger. Additionally, HIF-1 alpha may form a heterodimer with HIF-1 beta (Duan, et al. Circulation. 2005;111:2227-2232.).

Depending on the sample, treatment, etc. you may see either a band or a doublet.