Description
Human Hypoxia-inducible factor 1-alpha inhibitor (HIF1AN) ELISA Kit
The Human Hypoxia-Inducible Factor 1 Alpha Inhibitor (HIF1AN) ELISA Kit is specifically designed for the precise measurement of HIF1AN levels in human samples such as serum, plasma, and cell culture supernatants. This kit offers unparalleled sensitivity and specificity, ensuring accurate and consistent results for a diverse range of research applications.HIF1AN is a key regulator of the hypoxia response pathway, playing a critical role in cellular adaptation to low oxygen levels. Dysregulation of HIF1AN has been implicated in various diseases, including cancer, ischemic heart disease, and neurological disorders, highlighting its importance as a potential therapeutic target and a biomarker for disease progression.
With the Human HIF1AN ELISA Kit, researchers can gain valuable insights into the molecular mechanisms underlying hypoxia-induced signaling pathways and explore new avenues for drug discovery and personalized medicine approaches. Order your kit today and unlock the potential for groundbreaking discoveries in the field of hypoxia biology.
| Product Name: | Human Hypoxia-inducible factor 1-alpha inhibitor (HIF1AN) ELISA Kit |
| SKU: | HUEB0687 |
| Size: | 96T |
| Target: | Human Hypoxia-inducible factor 1-alpha inhibitor (HIF1AN) |
| Synonyms: | Factor inhibiting HIF-1, Hypoxia-inducible factor asparagine hydroxylase, FIH-1, FIH1 |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.312-20ng/mL |
| Sensitivity: | 0.12ng/mL |
| Intra CV: | 5.1% | ||||||||||||||||||||
| Inter CV: | 7.9% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Hydroxylates HIF-1 alpha at 'Asp-803' in the C-terminal transactivation domain (CAD). Functions as an oxygen sensor and, under normoxic conditions, the hydroxylation prevents interaction of HIF-1 with transcriptional coactivators including Cbp/p300-interacting transactivator. Involved in transcriptional repression through interaction with HIF1A, VHL and histone deacetylases. Hydroxylates specific Asn residues within ankyrin repeat domains (ARD) of NFKB1, NFKBIA, NOTCH1, ASB4, PPP1R12A and several other ARD-containing proteins. Also hydroxylates Asp and His residues within ARDs of ANK1 and TNKS2, respectively. Negatively regulates NOTCH1 activity, accelerating myogenic differentiation. Positively regulates ASB4 activity, promoting vascular differentiation. |
| Uniprot: | Q9NWT6 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Hypoxia-inducible factor 1-alpha inhibitor |
| Sub Unit: | Homodimer; homodimerization is essential for catalytic activity. Interacts with VHL and HIF1A. Part of a complex with VHL, HIF1A and HDAC1 or HDAC2 or HDAC3. Interacts with NFKB1 and NFKBIA. Interacts with NOTCH1, NOTCH2 and NOTCH3 but not with NOTCH4. Interacts with APBA3; binding inhibits HIF1AN binding to HIF1A. Interacts with TNKS2. Interacts with PPP1R12A. Interacts with ASB4 (By similarity). Interacts with UBE3A. Interacts with ANKS3. |
| Research Area: | Epigenetics |
| Subcellular Location: | Nucleus Cytoplasm Cytoplasm Perinuclear region Mainly cytoplasmic localization, but interaction with NOTCH1 results in nuclear localization and interaction with ABPA3 results in perinuclear localization in macrophages. |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | HIF1AN: Hydroxylates HIF-1 alpha at 'Asp-803' in the C-terminal transactivation domain (CAD). Functions as an oxygen sensor and, under normoxic conditions, the hydroxylation prevents interaction of HIF-1 with transcriptional coactivators including Cbp/p300- interacting transactivator. Involved in transcriptional repression through interaction with HIF1A, VHL and histone deacetylases. Hydroxylates specific Asn residues within ankyrin repeat domains (ARD) of NFKB1, NFKBIA, NOTCH1, ASB4, PPP1R12A and several other ARD-containing proteins. Also hydroxylates Asp and His residues within ARDs of ANK1 and TNKS2, respectively. Negatively regulates NOTCH1 activity, accelerating myogenic differentiation. Positively regulates ASB4 activity, promoting vascular differentiation. Homodimer; homodimerization is essential for catalytic activity. Interacts with VHL and HIF1A. Part of a complex with VHL, HIF1A and HDAC1 or HDAC2 or HDAC3. Interacts with NFKB1 and NFKBIA. Interacts with NOTCH1, NOTCH2 and NOTCH3 but not with NOTCH4. Interacts with APBA3; binding inhibits HIF1AN binding to HIF1A. Interacts with TNKS2. Interacts with PPP1R12A. Interacts with ASB4. |
| UniProt Protein Details: | Protein type:EC 1.14.11.30; EC 1.14.11.n4; Oxidoreductase Chromosomal Location of Human Ortholog: 10q24 Cellular Component: nucleoplasm; perinuclear region of cytoplasm; cytoplasm; cytosol; nucleus Molecular Function:NF-kappaB binding; protein binding; protein homodimerization activity; oxygen sensor activity; zinc ion binding; carboxylic acid binding; iron ion binding; oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors; Notch binding; cofactor binding Biological Process: peptidyl-aspartic acid hydroxylation; transcription, DNA-dependent; peptidyl-asparagine hydroxylation; negative regulation of Notch signaling pathway; positive regulation of myoblast differentiation |
| UniProt Code: | Q9NWT6 |
| NCBI GenInfo Identifier: | 32129605 |
| NCBI Gene ID: | 55662 |
| NCBI Accession: | Q9NWT6.2 |
| UniProt Secondary Accession: | Q9NWT6,Q5W147, Q969Q7, Q9NPV5, D3DR69, |
| UniProt Related Accession: | Q9NWT6 |
| Molecular Weight: | 40,285 Da |
| NCBI Full Name: | Hypoxia-inducible factor 1-alpha inhibitor |
| NCBI Synonym Full Names: | hypoxia inducible factor 1, alpha subunit inhibitor |
| NCBI Official Symbol: | HIF1AN |
| NCBI Official Synonym Symbols: | FIH1 |
| NCBI Protein Information: | hypoxia-inducible factor 1-alpha inhibitor; FIH-1; factor inhibiting HIF-1; factor inhibiting HIF1; hypoxia-inducible factor asparagine hydroxylase; peptide-aspartate beta-dioxygenase |
| UniProt Protein Name: | Hypoxia-inducible factor 1-alpha inhibitor |
| UniProt Synonym Protein Names: | Factor inhibiting HIF-1; FIH-1; Hypoxia-inducible factor asparagine hydroxylase |
| Protein Family: | Hypoxia-inducible factor 1-alpha inhibitor |
| UniProt Gene Name: | HIF1AN |
| UniProt Entry Name: | HIF1N_HUMAN |
| Component | Quantity (96 Assays) | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | -20°C |
| Lyophilized Standard | 2 | -20°C |
| Sample Diluent | 20ml | -20°C |
| Assay Diluent A | 10mL | -20°C |
| Assay Diluent B | 10mL | -20°C |
| Detection Reagent A | 120µL | -20°C |
| Detection Reagent B | 120µL | -20°C |
| Wash Buffer | 30mL | 4°C |
| Substrate | 10mL | 4°C |
| Stop Solution | 10mL | 4°C |
| Plate Sealer | 5 | - |
Other materials and equipment required:
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Incubator
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
| Step | |
| 1. | Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C. |
| 2. | Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform. |
| 3. | Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper. |
| 4. | Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C. |
| 5. | Repeat the wash process for five times as conducted in step 3. |
| 6. | Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction. |
| 7. | Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. |
| 8. | Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters. |
| 9. | After experiment, store all reagents according to the specified storage temperature respectively until their expiry. |
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
| Serum | If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. |
| Plasma | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit. |
| Urine & Cerebrospinal Fluid | Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid. |
| Cell culture supernatant | Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately. |
| Cell lysates | Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Tissue homogenates | The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C. |
| Tissue lysates | Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Breast Milk | Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles. |
