HIF-1 alpha detection in western blot

Protocol for inducing and detecting HIF-1 alpha expression.  

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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.

Induction of HIF-1 alpha by hypoxia

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.

  1. Set tissue culture incubator or chamber to an oxygen level of 2% or less through the regulated addition of nitrogen gas. Follow manufacturer instructions if you are using a hypoxic chamber.
  2. Grow your cells of choice to 70–80% confluency in the appropriate culture media.​
  3. Incubate cells in the hypoxic incubator or chamber.
  4. Take samples at multiple points.

    Maximum HIF-1 alpha induction takes place after ~ 4 hours (recommended incubation time: 2–8 hours).

  5. Aspirate media from cells and wash quickly with cold PBS.

    Work as quickly as possible as HIF-1 alpha is degraded in a normoxic environment.

  6. Lyse cells as described below.


Induction of HIF-1 alpha by CoCl2 (cobalt chloride)

  1. Grow your cells of choice to 70–80% confluency in the appropriate culture media.
  2. Immediately prior to use, prepare a 100 mM CoCl2 stock solution in PBS.
  3. Add 100–150 µM CoCl2 final solution directly to cell culture media. Mix well.
    Final concentrations of CoCl2 should be optimized for your specific cell type.
  4. Incubate cells under standard cell culture conditions (37ºC, 5% CO2) for 4–8 hours.
  5. Take samples at multiple points.
  6. Aspirate media from cells and wash quickly with cold PBS.
  7. Lyse cells as described below.


Cell lysis protocol

  1. Aspirate PBS.
  2. Add 1X Laemmli sample buffer (400 µL sample buffer for 10 cm dish).​
  3. Scrape cells with a cell scrapper until the sample buffer becomes viscous.
  4. Collect the buffer with lysed cells into a microcentrifuge tube.
  5. Sonicate sample until viscosity is reduced enough to allow you to pipette the lysate.
  6. Heat/boil sample at 95º​C for 5–10 minutes.​
  7. Load samples in SDS-PAGE.
  8. Run SDS-PAGE.

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/DFX (deferoxamine/desferrioxamine):

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 (dimethyloxalylglycine):

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.

HIF-1 alpha antibody [EP1215Y] (ab51608)

Figure 1. Example blot. Ramos cells (Burkitt's lymphoma cell line) were treated with CoCl2. Cells were lysed and 10 µ​g 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.

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