HIF Filter Plate Assay

Hypoxia-inducible factor (HIF) has been recognized primarily for its role in the maintenance of oxygen and energy homoeostasis. HIF-1 and HIF-2 are heterodimeric transcription factors composed of HIF-1 and HIF-2 , respectively, which dimerize with a constitutively expressed ß subunit and subsequently bind to hypoxia response elements in the promoters of target genes. The activity of HIF is regulated by a variety of stimuli, including changes in cellular oxygen concentration, growth factors and oncogenic activation. Signosis has developed HIF filter assay. Unlike conventional gel shift assays, HIF filter assay is a high throughput assay and it can analyze DNA binding activities of HIF in multiple samples simultaneously. Meanwhile, the assay can be set up for a few samples with less tedious than non-radioactive gel shift assay or much safer than 32P-based gel shift assay.

A. Benefits:

Signosis proprietary HIF filter Plate assay offers following benefits:

• Simple and quick -It is simpler and quicker than gel shift assay.

• High throughput screen- Multiple samples can be analyzed in one single assay.

• Cost-effective detection- The cost is lower than ELISA-based TF assays.

• Quantitative comparison The difference of two or more samples in TF activity can be quantitatively analyzed.

B. Principle of the technology

Signosis’ HIF filter plate assay is a plate-based analysis for monitoring the activity of HIF. In the assay, two plates – filter plate and hybridization plate – are needed. In the assay, pre-labeled HIF binding element is mixed with nuclear extract to allow formation of HIF/ probe complex. The filter is used to retain bound HIF probe and remove free probe. The bound HIF probe is then eluted from the filter and collected for plate hybridization. The captured probe is further detected with streptavidin-HRP. Luminescence is reported as relative light units (RLUs) on a microplate luminometer.