HIF-1 alpha (HIF1A)

The structure of the HIF-1 alpha protein

Figure 1: The structure of the HIF-1 alpha protein.

HIF-1 Alpha Introduction

Protein Function

HIF-1 alpha is a subunit of the HIF-1 protein, encoded by the HIF1A gene.
HIF-1 alpha is a master transcriptional regulator that enables cellular survival under hypoxic conditions.
It plays a essential role in embryonic vascularization, tumor angiogenesis and pathophysiology of ischemic disease.

Protein Characteristics

HIF-1 alpha is stable only at oxygen levels below 5%.
Under normoxic conditions, HIF-1 alpha has a very short half-life and may be nucleoplasmic degradation within short time.

Protein Expression

Under hypoxic conditions, HIF-1 alpha is expressed in most cell lines and tissues.
It is expressed with highest levels in the kidneys and heart.
Overexpression in the majority of common human cancers.

Protein Localization

Under normoxic conditions, HIF-1 alpha is expressed in the cytoplasm.
Under hypoxic conditions, HIF-1 alpha translocates to and is expressed in the nucleus.

HIF-1 alpha ICC Experimental Result Image Anti-HIF-1 alpha antibody [EPR16897] (ab179483)

Figure 2: HIF-1 alpha ICC Experimental Result Image Anti-HIF-1 alpha antibody [EPR16897] (ab179483). Green: HIF-1 alpha; Red: Tubulin; Blue: DAPI

Isoforms & Post-Translational Modifications

Isoforms:

Human (Q16665): Isoforms 1-3: 83-96 kDa (predicted)
Mouse (Q61221): Isoforms 1-2: 92-94 kDa (predicted)
Rat (O35800): 92 kDa (predicted)

Post-Translational Modifications:

Under normoxic (normal oxygen) conditions, HIF-1 alpha contains an oxygen-dependent degradation domain. It promotes interaction with VHL(von Hippel-Lindau), initiating rapid ubiquitination and subsequent proteasomal degradation.
Requires phosphorylation for DNA-binding.
Under hypoxic (low oxygen) conditions, HIF-1 alpha is not hydroxylated, allowing it to activate genes involved in hypoxic survival.

WB Experiment Tips

Precautions:

Samples typically need induction stimuli to detect HIF-1 alpha. Under normal oxygen conditions, HIF-1 alpha is largely undetectable. Inducing hypoxia in most cells and normal tissues is necessary to resolve no signal or weak signal issues.
It is recommended to treat samples with reagents such as DMOG, DFO, CoCl2, etc., to induce and maintain the expression of HIF-1 alpha (refer to positive controls).
After induction, HIF-1 alpha transiently expresses and is highly susceptible to degradation. It is recommended to treat samples with MG132 and add a proteasome inhibitor cocktail to the lysis buffer. Keep the sample preparation process on ice throughout, and proceed with WB immediately after preparation to prevent protein degradation from freezing.
If HIF-1 alpha signal is not detected with ab179483 antibody, it is strongly recommended to use the 1% SDS heat-mediated sample preparation method to enrich more protein. Click here to learn more about the 1% SDS heat-mediated sample preparation method.
Due to the existence of multiple forms of HIF-1 alpha, WB experiments may detect multiple bands or bands of sizes different from those predicted. To prevent loss of target signals, avoid trimming membranes.

Reference information:

40-80 kDa - Degraded forms of HIF-1 alpha
110-130 kDa- Post-translationally modified forms of HIF-1 alpha
~200 kDa - Heterodimers with HIF-1 beta

Positive Controls

Lysate of MCF-7 cells cultured under 0.5% oxygen for 24 hours.
Nuclear lysate of Hela cells treated with 0.5mM DFO for 24 hours.
Whole-cell lysate of Hela cells treated with 0.5mM CoCl2 for 6 hours.
Recombinant human HIF-1 alpha protein.

Negative Control

Most normal tissues and cells, excluding kidney and heart.

Example Results

WB-Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

Figure 3: WB-Anti-HIF-1 alpha antibody [EP1215Y] (ab51608)

Lane 1: MCF-7(normoxia)
Lane 2: MCF-7 treated with 0.5% oxygen for 24 hours

WB-Recombinant Anti-HIF-1 alpha Antibody [EPR16897] (ab179483)

Figure 4: WB-Recombinant Anti-HIF-1 alpha Antibody [EPR16897] (ab179483)

Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2: HeLa (Human cervix adenocarcinoma epithelial cell) treated with 0.5 mM CoCl2 for 6 hours whole cell lysates

Result Description: The different result is due to the lysates preparation method. The lysate in the left image is prepared by RIPA lysis method. The lysate in the right image is prepared by 1%SDS Hot lysis method. This antibody works better in 1%SDS Hot Lysates in WB.

Predicted band size: 92 kDa
Observed band size: 110 kDa

WB-Recombinant Anti-HIF-1 alpha Antibody [EPR16897] (ab179483)

Figure 5: WB-Recombinant Anti-HIF-1 alpha Antibody [EPR16897] (ab179483)

Lane 1: Untreated C6 (rat glial tumor glial cell), whole cell lysate
Lane 2: C6 treated with 400 µM CoCl2 and 20 µM MG-132 (ab141003) for 4 hours

Predicted band size: 92 kDa
Observed band size: 110 kDa

Result Description: The expression of HIF-1 alpha is induced by CoCl2 and maintained by MG-132 treatment (PMID: 15836611)

Key control points

In the experiment, in addition to paying attention to routine issues, special attention should be paid to the following key control points:

Sample preparation:

Sonicate cell samples to release your target protein into solution and obtain a higher yield.
Add a protease inhibitor cocktail to prevent degradation of target proteins.
Keep samples on ice throughout the entire sample preparation process.
Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis:

Load at least 50 μg of total protein from cell lysate or tissue homogenate.

Transfer:

It is recommended not to cut the membrane and keep the entire membrane for antibody incubation
It is recommended to stain the membrane with Ponceau S after the transfer to confirm the success of the transfer.

Antibody incubation:

Select a suitable antibody working concentration according to the product datasheet.
It is not recommended to reuse antibodies and to always use fresh antibody preparations.