For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome
Protocol for inducing and detecting HIF-1 alpha expression.
HIF-1 alpha is a major global regulator of hypoxic response, and it is rapidly degraded in oxygenated cells. Degradation can occur within the first five minutes of exposure to normoxia, even after cells have been growing for long period in hypoxia.
Here we provide a protocol to help you induce HIF-1 alpha expression in culture cells and identify the protein by western blot.
For this step you need a tissue culture incubator capable of monitoring and regulating oxygen levels. Alternatively, you can use a hypoxic chamber on a general tissue culture incubator.
Maximum HIF-1 alpha induction takes place after ~ 4 hours (recommended incubation time: 2–8 hours).
Work as quickly as possible as HIF-1 alpha is degraded in a normoxic environment.
This lysis method is designed to minimize protein degradation on the sample. However, it does not allow you to quantify the amount of protein in the sample.
If you need to quantify protein, we recommend lysing your cells in RIPA buffer containing protease inhibitors. Because HIF-1 alpha is rapidly degraded in normoxia conditions, we recommend using HIF-1 stabilizers to inhibit degradation. The most used are
DFO and DFX are iron chelators taht will sequester iron present in cells. HIF-1 alpha is known to degrade in the presence of oxygen and iron. When iron or oxygen levels are low, prolyl-4-hydroxylase domain (PHD) enzymes cannot hydroxylate HIF-1 alpha.
DMOG is an ester of N-oxalylglycine that inhibits the PHD enzymes that regulate HIF-1 alpha stability. DMOG stabilizes HIF-1 alpha expression at concentrations between 0.1–1 mmol/L.
Under hypoxic conditions, HIF-1 alpha is stabilized and translocates to the nucleus. Detection of HIF-1 alpha using nuclear lysates will increase the signal.
Figure 1. Example blot. Ramos cells (Burkitt's lymphoma cell line) were treated with CoCl2. Cells were lysed and 10 µg was loaded into a 10% SDS-PAGE. HIF-1 alpha was detected with a rabbit monoclonal anti-HIF-1 alpha antibody [EP1215Y] (ab51608) at 1:1,000 dilution.