Human HIF-1alpha Transcription Factor Activity Assay by RayBiotech Inc.
Manufacturer RayBiotech Inc. | Model: TFEH-HIF1a | Available Worldwide
RayBio® Human HIF-1? Transcription Factor Activity Assay. This assay uses a dsDNA coated plate with canonical HIF-1? binding sequences to semi-quantitatively detect active HIF-1? in lysates or nuclear extracts.
Human HIF-1alpha Transcription Factor Activity Assay
Introduction
The changes of environmental oxygen levels induce a number of metabolic changes with rapid and profound consequences for cell physiology. To regulate oxygen homeostasis, the transcription factor hypoxia-inducible factor (HIF) regulates the expression of up to 50 adaptive genes by binding to hypoxia response elements (HRE) in their promoters. HIF consists of two subunits: oxygen-regulated α-subunit (HIF-1α) and constitutively expressed nuclear β-subunit (HIF-1β). In normoxia, HIF-1α is hydroxylated by oxygen-sensitive prolyl hydroxylases (PHDs) at Pro-402 and/or Pro-564 and asparaginyl hydroxylase FIH at Asn-80. Modified HIF-1α is subsequently destined to be degrade or prevented from the recruitment of the transcriptional coactivators p300 and CBP, resulting in low protein levels within cellular nuclear. Due to high availability of oxygen in hypoxia, however, hydroxylase activity decreases, thus enabling HIF-1a to accumulate and translocate to the nucleus where it forms transcriptional complex with the HIF1b/ARNT subunit and binds to HRE sequence to initiate gene expression.
Product Features
- Specific transcription factor-DNA binding assay
- Perfect alternative to EMSA
- Easy to perform in an ELISA format
- Non-radioactive assay
- High throughput (96-well plate format)
- Assay can be completed within 5 hours
Specificity
The olionucleotide/antibody pair provided in this kit recognizes human HIF-1α in whole lysates and nuclear extracts.
Target Protein Information
Synonyms
Hypoxia-inducible factor 1-alpha (HIF-1-alpha) (HIF1-alpha) (ARNT-interacting protein) (Basic-helix-loop-helix-PAS protein MOP1) (Class E basic helix-loop-helix protein 78) (bHLHe78) (Member of PAS protein 1) (PAS domain-containing protein 8)
Accession Number
Gene Names
HIF1A BHLHE78 MOP1 PASD8
Gene ID
3091
Species Detected
Human
Application Notes
Kit Components
- 96-well Strip Microplate pre-coated with DNA probes
- DNA Binding Buffer
- Positive Control Sample
- Specific Competitor DNA probe
- Non-specific Competitor DNA probe
- Assay Reagent
- DTT
- Wash Buffer
- Primary Antibody
- HRP-conjugated Secondary Antibody
- TMB One-Step Substrate Reagent
- Stop Solution
Other Materials Required
- Distilled or deionized water
- 100 ml and 1 liter graduated cylinders
- Tubes to prepare sample dilutions
- Absorbent paper
- Precision pipettes to deliver 2 µl to 1 ml volumes
- Adjustable 1-25 ml pipettes for reagent preparation
- Benchtop rocker or shaker
- Microplate reader capable of measuring absorbance at 450 nm
Protocol Outline
- Prepare all reagents and samples as instructed in the manual.
- Add 100 µl of sample or positive control to each well.
- Incubate 2 h at RT or O/N at 4 °C.
- Add 100 µl of prepared primary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of prepared HRP-secondary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of TMB One-Step Substrate Reagent to each well.
- Incubate 30 min at RT.
- Add 50 µl of Stop Solution to each well.
- Read at 450 nm immediately.
Storage/Stability
Upon receipt, the positive control should be removed and stored at -20° or -80°C. The remainder of the kit can be stored for up to 6 months at 2-8°C from the date of shipment. Opened Microplate Wells or reagents may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack, reseal along entire edge.
Note: The kit can be used within one year if the whole kit is stored at -20°C upon receipt. Avoid repeated freeze-thaw cycles.
Note: The kit can be used within one year if the whole kit is stored at -20°C upon receipt. Avoid repeated freeze-thaw cycles.
Product Overview
Human HIF-1alpha Transcription Factor Activity Assay
Manufacturer RayBiotech Inc. | Available Worldwide
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