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Western blot coomassie blue stain

HIF-1 alpha target tips

​​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.

​​Overview

​Sample preparation

Electrophoresis

Transferring

Maximizing signal

Recommended controls


Protein function, expression, and isoforms

Protein function

  • HIF-1 alpha functions as a master transcriptional regulator of the adaptive response to hypoxia
  • Under hypoxic conditions, it can activate over 40 genes, whose protein products increase oxygen delivery or facilitate metabolic adaptations to hypoxia

Expression

  • HIF-1 alpha is expressed in most cell lines and tissues (under hypoxic conditions) 
  • Its highest levels are found in kidney and heart tissues, and it is also overexpressed in the majority of human cancers
  • Under normoxic conditions, HIF-1 alpha is largely undetectable. Hypoxia needs to be induced in most cells and normal tissues in order to isolate and detect HIF-1 alpha.

Isoforms

  • Isoform 1: 93 kDa (predicted) 
  • Isoform 2: 83 kDa (predicted) 
  • Isoform 3: 96 kDa (predicted)

Explore HIF-1 alpha's role in depth with our interactive pathways

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Western Blot tips

HIF-1 alpha can be a challenging target to work with in western blot.

HIF-1 alpha is only stabilized at O2 concentrations 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, you might not see any bands on western blot.

Note: the observed band size of HIF-1 alpha may not be the same as predicted in western blot due to the different forms of HIF-1 alpha, including:

  • 40-80 kDa - degraded HIF-1 alpha
  • 110-130 kDa - post-translationally modified HIF-1 alpha
  • ~200 kDa - heterodimer with HIF-1 beta

Sample preparation
  • Add a protease inhibitor such as MG132, which can stabilize HIF-1 alpha and prevent protein degradation 1.
  • CoCl2 or deferoxamine (DFO) can be used to induce hypoxia 2​.

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Electrophoresis

  • Run samples in 8% or lower separating gel.
  • Load at least 50 μg 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.

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Transferring
  • We recommend adding SDS to a final concentration of 0.1% in the transfer buffer.
  • Wash PVDF membrane to remove methanol completely.
  • Determine whether the transfer is successful by visualization of proteins in membranes using Ponceau S.

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Maximizing signal
  • Hypoxic chambers may be used to incubate samples overnight at low oxygen pressure to increase HIF-1 alpha levels.
  • Cells should be lysed as quickly as possible (within 2 minutes) if removed from hypoxia.
  • Overnight incubation at 4°C with the primary antibody can also help.

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Recommended controls


Positive control: Hypoxic samples, e.g.  Deferoxamine (DFO) treated HeLa extracts (ab180880).

Negative control: Most normal tissues or cells (other than kidney or heart) should work as a negative control, as HIF-1 alpha will degrade when taken out of hypoxic conditions.

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References

  1. ​Moroz E et. al (2009). Real-time imaging of HIF-1alpha stabilization and degradation. PLoS One; 4(4):e5077
  2. Wu D, Yotnda P. (2011) Induction and testing of hypoxia in cell culture. J Vis Exp. 12;(54):2899

View these tips as a PDF: 

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