The olionucleotide/antibody pair provided in this kit recognizes human HIF-1? in whole lysates and nuclear extracts.
Number of Targets Detected:
Compatible Sample Types:
- Cell Lysates
- Nuclear Extracts
Method of Detection:
1, 2, or 5 x 96-Well Strip Microplate Kit
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 Ã±-subunit (HIF-1Ã±) and constitutively expressed nuclear Ã²-subunit (HIF-1Ã²). In normoxia, HIF-1Ã± is hydroxylated by oxygen-sensitive prolyl hydroxylases (PHDs) at Pro-402 and/or Pro-564 and asparaginyl hydroxylase FIH at Asn-80. Modified HIF-1Ã± 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.
- 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
- 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
- Wash Buffer
- Primary Antibody
- HRP-conjugated Secondary Antibody
- Antibody Diluent Buffer
- 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
- Microplate reader capable of measuring absorbance at 450 nm
- 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.
Transcription factor activity assay of HIF-1Ã± from nuclear extracts of HeLa cells or HeLa cells treated with DMOG (1mM) for 4 hr. A. Western-blot result of HIF-1Ã± from cytoplasm and nuclear fractions. B. Transcription factor activity assay of HIF-1Ã± from nuclear fractions with the RayBio® HIF-1Ã± Transcription Factor Activity Assay Kit.
Transcription factor activity assay of HIF-1Ã± from nuclear extracts of HeLa cells or HeLa cells treated with DMOG (1mM) for 4 hr with the specific competitor or non-specific competitor. The result shows specific binding of HIF-1Ã± to the HRE binding site.
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.