TransAM^® HIF-1

HIF-1 Transcription Factor Info

The transcription factor Hypoxia-Inducible Factor 1 (HIF-1) is one of the key regulators of oxygen homeostasis. It regulates the physiological responses to low oxygen levels (hypoxia) and the pathophysiology of heart attack, cancer, stroke and chronic lung disease. HIF-1 is a heterodimeric protein that consists of two subunits, HIF-1α and HIF-1β Whereas HIF-1β is constitutively expressed, the expression of HIF-1α is induced by oxygen concentrations below 6%. HIF heterodimers bind to the hypoxia response element (HRE), a 5´-RCGTG-3´ consensus sequence. Several dozen HIF-1-regulated genes have been identified so far, including genes coding for proteins involved in angiogenesis, energy metabolism, erythropoiesis, cell proliferation and viability, vascular remodeling and vasomotor responses.

Figure 1: Measurement of HIF-1α.

Nuclear extracts were isolated from COS-7 cells using the Nuclear Extract Kit. The cells were either treated for 20 hours with 0.15 mM CoCl₂ or were untreated. Increasing amounts of nuclear extract are assayed for HIF-1α activation using the TransAM HIF-1 Kit.

The TransAM^® transcription factor ELISA advantage

Historically, transcription factor studies have been conducted using gelshift, Western blot and reporter plasmid transfections, which are time-consuming, do not allow for high-throughput and provide only semi-quantitative results. TransAM assays are up to 100 times more sensitive than gelshift techniques, and can be completed in less than 5 hours. Because TransAM is an ELISA-based assay*, there is no radioactivity, and the high-throughput stripwell format enables simultaneous screening of 1-96 samples. Inconsistencies due to variable reporter plasmid transfections are eliminated, along with the need to construct stable cell lines.

Why use TransAM^® transcription factor ELISAs?

• Up to 100-fold more sensitive than gelshift assays
• Eliminates the use of radioactivity and the need to run gels
• Results in less than five hours
• Colorimetric readout enables easy, quantitative analysis with spectrophotometry at 450 nm
• 96-stripwell format enables both high and low throughput

How TransAM^® transcription factor ELISAs work

The TransAM format is perfect for assaying transcription factor binding to a consensus-binding site. TransAM Kits contain a 96-stripwell plate to which the consensus-binding site oligo has been immobilized. Activated nuclear extract is added to each well and the transcription factor of interest binds specifically to this bound oligonucleotide. A primary antibody specific for an epitope on the bound and active form of the transcription factor is then added followed by subsequent incubation with secondary antibody and Developing Solution to provide an easily quantified, sensitive colorimetric readout (Figure 1).

Figure 1: Flow chart of the TransAM process.

Activated transcription factor in the cell extract binds to the consensus-binding site on the oligo immobilized in the well. Incubation with the supplied primary and secondary antibodies specifically quantifies the amount of activated transcription factor.

* Technology covered under EAT-filed patents and licensed to Active Motif. Use of TransAM in NFκB-related drug discovery may be covered under U.S. Patent No. 6,150,090 and require a license from Ariad Pharmaceuticals (Cambridge, MA, USA).

Contents & Storage

Each TransAM HIF-1 Kit contains HIF-1α primary antibody, HRP-conjugated secondary antibody, HIF-1 wild-type and mutated oligonucleotides, COS-7 nuclear extract (positive control), Dithiothreitol, Protease Inhibitor Cocktail, Herring Sperm DNA, Lysis Buffer, Binding Buffer, 10X Washing Buffer, 10X Antibody Binding Buffer, Developing Solution, Stop Solution and one or five HIF-1 96-well assay plate(s) with plate sealer(s). Reagent storage conditions vary from room temperature to -80°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.