For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome
If you continue without changing your cookie settings, we'll assume you’re happy with this.
Hypoxia-inducible factor 1 alpha (HIF-1 alpha) is a subunit of a heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1) that is encoded by the HIF1A gene. HIF-1 alpha has an essential role in response to cellular hypoxia and is involved in angiogenesis and erythropoiesis processes as well as cell proliferation and survival.
HiF-1 alpha can be a challenging target to work with in western blot.
HIF-1 alpha is only stabilized at O2 concentration below 5%. Under normoxic conditions HIF-1 alpha has a short half-life and may be degraded within 5-8 minutes in both nuclear and cytoplasmic compartments. Therefore, proper sample preparation is critical for western blot success. If care hasn’t been taken with sample preparation, no bands may be seen on your blot. The observed band size of HIF-1 alpha may not be the same as predicted MWs in western blot due to the different forms of HIF-1 alpha.
|Sample preparation||Add adequate protease inhibitors (or phosphatase inhibitors for proteins modified by phosphorylation) to avoid target protein degradation.|
|Ultrasonicate samples to enrich more target proteins.|
|Keep samples on ice during the whole WB process.|
|Perform a Bradford assay, a Lowry assay or a bicinchoninic acid (BCA) assay to determine the protein concentration.|
|Electrophoresis||For large proteins (the MW of target protein >100 kDa), be sure to run samples in 8% or lower separating gel.|
|Load at least 50μg total protein per lane.|
|We strongly recommend the use of a positive control lysate when setting up a new experiment; this will give you immediate confidence in the protocol.|
|Transferring||It is preferred to add SDS to a final concentration of 0.1% in the transfer buffer for large proteins.|
|Wash PVDF membrane to remove methanol completely|
|To determine if the transfer is successful by visualization of proteins in membranes using Ponceau S.|
|Maximizing signal||Hypoxic chambers may be used to incubate samples overnight at low oxygen pressure to induce HIF-1 alpha levels.|
|Cells should be lysed as quickly (within 2 mins) as possible if removed from hypoxia.|
|Use positive control samples such as nuclear lysates of DFO or CoCl2 treated cells.|
|Overnight incubation at 4°C with the primary antibody can also help.|
The observed band size of HIF-1 alpha is not exactly as predicted 93 kDa in WB due to the different forms of HIF-1 alpha as below:
HeLa-DFO treated nuclear extracts (for stronger signal)
Most normal tissues or cells (other than kidney or heart)