HIF-1 alpha Cellular Assay Kit

HIF-1α assay for measuring this master transcriptional regulator of cellular and developmental response to hypoxia

HIF-1α is one of the 2 subunits of the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF1 acts as a master regulator of cellular and systemic response to hypoxia by activating the translation of genes involved in energy metabolism, angiogenesis, and apoptosis, among others. High levels of HIF 1 have been associated with aggressive tumor progression, and thus it has been investigated as a predictive and prognostic marker for resistance to radiation treatment and chemotherapy.

HIF-1α is also strongly linked to diabetes through its involvement in the glucose uptake of target cells. It has also been implicated in diabetes complications, for example diabetic nephropathy or diabetic wound repair.

Assay principle

HTRF® - THE HOMOGENEOUS CELL-BASED SANDWICH IMMUNOASSAY

The HIF-1α HTRF assay is a sandwich immunoassay involving two specific anti-human anti-HIF-1α antibodies, respectively labelled with Europium cryptate (donor) and d2 (acceptor). The intensity of the signal is proportional to the concentration of anti-HIF-1α present in the sample. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only small sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

After cell lysis, HIF-1α can be detected using the HTRF HIF-1α cellular assay kit reagents and most TR-FRET multimode plate readers.

 

A SIMPLER, MORE FLEXIBLE ASSAY PROTOCOL - ADAPTED TO YOUR APPLICATIONS

TWO-PLATE ASSAY PROTOCOL

Cells are plated, stimulated and next lysed in the same 96-well culture plate. Lysates are then transferred to the assay plate for the detection of HIF-1α by HTRF® reagents. This protocol enables the cells' viability and confluence to be monitored. The antibodies labelled with HTRF fluorophores may be pre-mixed and added in a single dispensing step, to further streamline the assay procedure. The assay detection can be run in 96- to 384-well plates by simply resizing each addition volume proportionally.

Product performance

1- HTRF ASSAY COMPARED TO WESTERN BLOT USING HIF-1Α CELLULAR ASSAY ON HUMAN HELA CELLS

Human HeLa cervical cancer cells were seeded in a T175 flask in complete culture medium, and incubated for 2 days at 37°C, 5% CO2 until 80% confluency was reached. After treatment (6 hours, 10 µM MG-132, 1 mM DMOG), the cells were lysed with 3 mL of supplemented lysis buffer#4 for 30min at RT under gentle shaking. Soluble supernatants were collected after a 10 minute centrifugation.

Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF HIF-1α detection reagents. Equal amounts of lysates were used for a side by side comparison of Western Blot and HTRF®.

Using HTRF® HIF-1α, just 3120 cells were sufficient for minimal signal detection while 25,000 cells were needed for a Western Blot signal. The HTRF® assay is 8-fold more sensitive than the Western Blot.

2- STABILISING EFFECT OF A CHEMICAL HYDROXYLASE INHIBITOR AND OF A PROTEASOME INHIBITOR ON HIF-1Α

Murine embryonic fibroblasts NIH-3T37 were plated at 100,000 cells/ well in a 96 well plate. After treatment for 6 hours at 37°C (in triplicate) with increasing concentrations of the hydroxylase inhibitor DMOG to prevent HIF-1α hydroxylation, or with increasing concentrations of the proteasome inhibitor MG-132 to inhibit HIF-1α degradation, the medium was removed and the cells were lysed with 50 µL of supplemented lysis buffer #4 for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF HIF-1α were added. The HTRF signal was recorded after an overnight incubation.

3- HIF-1Α AND PHOSPHO-ERK1/2 UPREGULATION IN HUMAN A431 CANCER CELLS USING EGF GROWTH FACTOR

Human A431 epidermoid carcinoma cells were plated at 100,000 cells/ well in a 96 well plate. After a preincubation with 1mM DFO for 30 minutes to prevent the degradation of HIF-1α, the cells were treated for 6 h at 37°C with increasing concentrations of EGF to activate the ERK pathway and the translation of HIF-1α. The medium was then removed and the cells were lysed with 50 µL of supplemented lysis buffer#4 for 30min at RT under gentle shaking.

16 µL of lysate were then transferred into 2 different wells of a 384-sv plate for measurement in parallel. Phosphorylated ERK ½ and HIF-1α, and 4 µL of HTRF HIF-1α or phospho-ERK detection reagents were then added and the signal was recorded after an overnight incubation at RT.

4- HIF-1Α UPREGULATION IN HUMAN MCF-7 CANCER CELLS USING IGF-1 GROWTH FACTOR

Human MCF-7 epidermoid carcinoma cells were plated at 50,000 cells/ well in a 96 well plate. After a preincubation with 1mM DFO for 30 minutes to prevent the degradation of HIF-1α, the cells were treated for 6 h at 37°C with increasing concentrations of IG-1F to activate the AKT pathway and the translation of HIF-1α. The medium was then removed and the cells were lysed with 50 µL of supplemented lysis buffer#4 for 30min at RT under gentle shaking.

16 µL of lysate were transferred into a 384-well low volume white microplate for detection  and 4 µL of the HTRF HIF-1α detection reagents were added. The HTRF signal was then recorded after an overnight incubation.

5- HIF-1Α DOWNREGULATION IN HUMAN MCF-7 CANCER CELLS USING A LY294002 PI3K INHIBITOR

Human MCF-7 epidermoid carcinoma cells were plated at 50,000 cells/ well in a 96 well plate. After a preincubation with increasing concentrations of 1mM DFO for 30 minutes to prevent the degradation of HIF-1α, the cells were treated for 6 h at 37°C with increasing concentrations of the PI3K inhibitor LY294002 for 30 minutes (in triplicate) to inhibit the PI3K/AKT pathway. The cells were then activated with 10 nM IGF-1 and 1mM DFO. Next, the medium was removed and the cells were lysed with 50 µL of supplemented lysis buffer#4 for 30min at RT under gentle shaking.

16 µL of lysate were then transferred into 2 different wells of a 384-sv plate for measurement in parallel. Phosphorylated AKT, HIF-1α and 4 µL of HTRF HIF-1α or phospho-AKT Ser473 detection reagents were then added. The signal was recorded after an overnight incubation at RT.

Simplified pathway

Hypoxia-inducible factor 1-alpha, also known as HIF-1α, is a subunit of the heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1).

HIF1 consists of a constitutively expressed HIF-1β subunit (87 kDa) and an O2-regulated HIF-1α subunit (120 kDa) which contains an O2-dependent degradation domain (ODD).

  • Under normoxia, HIF-1α is hydroxylated on its ODD domain by O2-activated prolyl hydroxylases (PHDs), leading to its rapid ubiquitination and subsequent degradation by the proteasome (half-life < 5 min).
  • Under hypoxia, PHD activity is inhibited and HIF-1α accumulates in the cytosol, translocates to the nucleus, heterodimerizes with HIF-1β, and is activated. The complex activates the transcription of more than 100 target genes that facilitate responses to the hypoxic environment by encoding proteins involved in angiogenesis (e.g. VEGF), erythropoiesis (e.g. EPO), anaerobic glucose metabolism, cell proliferation/survival, and inflammation.

Hypoxic conditions can be mimicked by chemical hydroxylase inhibitors such as Dimethyloxalylglycine (DMOG), Deferoxamine (DFO) and Cobalt chloride (CoCl2), which inhibit HIF-1α degradation and lead to its stabilization. In addition, HIF-1α translation can be induced in a hypoxia-independent manner by growth factors through the AKT/mTOR and the MEK/ERK pathways.

  • HIF-1α can also be upregulated by inflammatory mediators.
  • In tumor cells, HIF-1α is overexpressed, which promotes cancer progression and metastasis.

In diabetes, hyperglycemia leads to a defect in the stabilization of HIF-1α under hypoxia, resulting in poor cell and tissue responses to low oxygen and chronic complications.

 

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
HIF-1 alpha kit - 500 tests64HIFPEG
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HIF-1 alpha kit - 10,000 tests64HIFPEH
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Companion products

DescriptionCat. noProduct insertMSDS
HIF-1 alpha kit control lysate64HIFTDA
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Phospho-total protein lysis buffer #4 - 130 mL64KL4FDF

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