Western Blot Technique for detection of HIF-1 Alpha

This article describes the steps for the detection of HIF-1 alpha expression using western blot. HIF-1 alpha is a protein that is expressed in all cells and regulates the response of cells to hypoxia that breaks down rapidly - within a few minutes - when the cells are exposed to oxygen. This phenomenon is observed even after long periods of cell growth in a hypoxic setting.

The protocol provided here shows how to bring about HIF-1 alpha expression within cultured cells and the means to identify it using western blot techniques.

Inducing HIF-1 Alpha by Hypoxia

Since hypoxia is the primary inducer of this HIF-1 alpha, a tissue culture incubator must be used to set and monitor oxygen levels as desired. If this is not available, an ordinary tissue culture incubator can be used, along with a hypoxic chamber.

  1. The incubator or hypoxic chamber is set to contain oxygen at 2% or less using nitrogen gas as the additive. The instructions in the manual must be followed if a hypoxic chamber has been selected.
  2. The cells already chosen are now grown within the appropriate culture media until confluency of 70-80% is seen.
  3. The cells are incubated in the chosen setting.
  4. Samples are taken from several points.

Note: Incubation should proceed for 2-8 hours. HIF-1 alpha induction peaks after about 4 hours have elapsed.

  1. The media are aspirated from the cells and they are rapidly washed using cold PBS. Quick work is required because of the rapid degradation of HIF-1 alpha that is seen when the oxygen level is normal.
  2. The cells are lysed.

Inducing HIF-1 Alpha by Cobalt Chloride

Here the protocol is as follows:

  1. The cells are grown in the selected culture medium to a confluency of 70-80%.
  2. 100 mM stock solution of CoCl2 in PBS is prepared immediately before use.
  3. 100-150 µM of this solution is added to the cell culture media before being mixed thoroughly.

The final concentration of cobalt chloride is to be determined as is appropriate for the type of cell used.

  1. The cells are incubated at 37 oC with 5% carbon dioxide, standard cell culture conditions, for 4-8 hours.
  2. Samples are taken from several points.
  3. Media is aspirated from the cells, which are then quickly washed using cold PBS.
  4. The cells are lysed.

Lysis of Cells

The cell lysis protocol is as follows:

  1. PBS is aspirated.
  2. 1X Loemmli sample buffer is added to the culture dishes at the rate of 400 µL per 10 cm dish.
  3. The cell are scraped using a cell scraper until the point at which the sample buffer turns viscous.
  4. The buffer, along with the lysed cells, is collected into a microcentrifuge tube.
  5. The sample is sonicated to reduce the viscosity up to the point where the lysed solution can be pipetted.
  6. The sample is boiled or heated to 95 oC for 5-10 minutes.
  7. The samples are loaded in SDS-PAGE.
  8. The SDS-PAGE is run.

This method of lysis is planned so that protein degradation in the sample will be reduced to the minimum. The drawback is that protein quantification is not possible.

Thus, if the quantity of protein needs to be measured, the lysis should be carried out using RIPA buffer produced with protease inhibitors. The use of HIF-1 alpha stabilizers is advised to inhibit the rapid degradation of HIF-1 alpha that occurs when cells are oxygenated. These stabilizers include:

  • DFO/DFX (deferoxamine/desferrioxamine): these compounds chelate iron. That is, they entrap the iron present within the cells. Oxygen and iron promote HIF-1 alpha degradation. At low levels of oxygen and iron, PHD (prolyl-4-hydroxylase domain) enzymes cannot produce HIF-1 alpha hydroxylation.
  • DMOG (dimethyloxalylglycine): this is an esterified form of N-oxalylglycine which inhibits PHD enzymes and thus stabilize HIF-1 alpha when added at concentrations of 0.1-1 mmol/L.

When the cell is hypoxic, HIF-1 alpha stabilization occurs and it enters the nucleus. When nuclear lysates are used to detect HIF-1 alpha the signal will therefore be stronger.

Example blot. Ramos cells (Burkitt

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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Last updated: Apr 1, 2019 at 6:22 AM


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