HIF-1 alpha ELISA Kit (HUFI00398)
- SKU:
- HUFI00398
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- Q16665
- Sensitivity:
- 0.094ng/ml
- Range:
- 0.156-10ng/ml
- ELISA Type:
- Sandwich
- Synonyms:
- HIF-1alpha, HIF1A, ARNT-interacting protein, Basic-helix-loop-helix-PAS protein MOP1, BHLHE78, Class E basic helix-loop-helix protein 78, HIF-1-alpha, HIF1-alpha, hypoxia-inducible factor 1-alpha, Member of PAS protein 1, member of PAS superfamily 1,
- Reactivity:
- Human
Description
HIF-1 alpha ELISA
HIF1A (Hypoxia Inducible Factor 1 Subunit Alpha) is a protein that is expressed in response to low oxygen levels. It has been shown to play a role in tumorigenesis and metastasis. HIF1A is involved in the process of angiogenesis, which is the formation of new blood vessels. HIF1A also plays a role in the regulation of energy metabolism. Diseases associated with HIF1A include Hypoxia and Retinal Ischemia. Among its related pathways are Signaling by PTK6 and CDK-mediated phosphorylation and removal of Cdc6.
Key Features
| Save Time | Pre-coated 96 well plate | |
| Quick Start | Kit includes all necessary reagents | |
| Publication Ready | Reproducible and reliable results |
Overview
| Product Name: | HIF-1 alpha ELISA Kit |
| Product Code: | HUFI00398 |
| Size: | 96 Assays |
| Alias: | HIF-1alpha, HIF1A, ARNT-interacting protein, Basic-helix-loop-helix-PAS protein MOP1, BHLHE78, Class E basic helix-loop-helix protein 78, HIF-1-alpha, HIF1-alpha, hypoxia-inducible factor 1-alpha, Member of PAS protein 1, member of PAS superfamily 1, PAS domain-containing protein 8 |
| Detection Method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human HIF1A concentrations in serum plasma and other biological fluids. |
| Sensitivity: | 0.094ng/ml |
| Range: | 0.156-10ng/ml |
| Storage: | 4°C for 6 months |
| Note: | For Research Use Only |
Additional Information
| Recovery: | Matrices listed below were spiked with certain level of Human HIF1A and the recovery rates were calculated by comparing the measured value to the expected amount of Human HIF1A in samples.
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| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human HIF1A and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
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| CV(%): | Intra-Assay: CV<8% |
Kit Components
| Component | Quantity | Storage |
| ELISA Microplate (Dismountable) | 8x12 strips | 4°C for 6 months |
| Lyophilized Standard | 2 | 4°C/ -20°C |
| Sample/Standard Dlution Buffer | 20ml | 4°C |
| Biotin-labeled Antibody (Concentrated) | 120ul | 4°C (Protection from light) |
| Antibody Dilution Buffer | 10ml | 4°C |
| HRP-Streptavidin Conjugate (SABC) | 120ul | 4°C (Protect from light) |
| SABC Dilution Buffer | 10ml | 4°C |
| TMB Substrate | 10ml | 4°C (Protection from light) |
| Stop Solution | 10ml | 4°C |
| Wash Buffer (25X) | 30ml | 4°C |
| Plate Sealer | 5 | - |
Other materials 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
Protein Information
| Uniprot: | |
| UniProt Protein Function: | HIF1A: a master transcriptional regulator of the adaptive response to hypoxia. Under hypoxic conditions, activates the transcription of over 40 genes, including erythropoietin, glucose transporters, glycolytic enzymes, vascular endothelial growth factor, HILPDA, and other genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia. |
| UniProt Code: | |
| NCBI GenInfo Identifier: | |
| NCBI Gene ID: | |
| NCBI Accession: | |
| UniProt Related Accession: | |
| Molecular Weight: | 95,634 Da |
Protocol
*Note: Protocols are specific to each batch/lot. For the exact instructions please follow the protocol included in your kit.
Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.
| Step | Procedure |
| 1. | Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells! |
| 2. | Aliquot 0.1ml standard solutions into the standard wells. |
| 3. | Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well. |
| 4. | Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells. |
| 5. | Seal the plate with a cover and incubate at 37 °C for 90 min. |
| 6. | Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2. |
| 7. | Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall. |
| 8. | Seal the plate with a cover and incubate at 37°C for 60 min. |
| 9. | Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash. |
| 10. | Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min. |
| 11. | Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min. |
| 12. | Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color. |
| 13. | Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately. |
| 14. | Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution. |
Sample Type
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. |
HIF-1 alpha Background
HIF-1 alpha stands for Hypoxia-Inducible Factor 1 alpha. It is a protein that plays a critical role in cellular responses to low oxygen levels, also known as hypoxia. HIF-1 alpha is a subunit of the HIF-1 transcription factor, which regulates the expression of genes involved in oxygen homeostasis and adaptation to hypoxic conditions.
Regulation and Function of HIF-1 alpha
In the presence of normal oxygen levels, HIF-1 alpha is rapidly degraded by the action of prolyl hydroxylase enzymes. However, under hypoxic conditions, the degradation of HIF-1 alpha is suppressed, allowing it to accumulate and translocate to the cell nucleus. In the nucleus, HIF-1 alpha forms a complex with another subunit, HIF-1 beta, to create an active HIF-1 transcription factor.
Role of HIF-1 alpha in Gene Regulation
The HIF-1 transcription factor binds to specific DNA sequences called hypoxia response elements (HREs) in the promoter regions of target genes. This binding leads to the upregulation of various genes involved in oxygen delivery, energy metabolism, angiogenesis, and cell survival. These genes include those encoding erythropoietin (EPO), vascular endothelial growth factor (VEGF), glucose transporters (GLUTs), and glycolytic enzymes.
Activation and Factors Influencing HIF-1 alpha
The activation of HIF-1 alpha is not only triggered by hypoxia but can also be influenced by other factors such as growth factors, cytokines, and certain oncogenic mutations. Dysregulation of HIF-1 alpha has been associated with a variety of diseases, including cancer, cardiovascular disorders, and neurodegenerative conditions.
HIF-1 alpha FAQs
What is the HIF-1 alpha ELISA Kit?
The HIF-1 alpha ELISA Kit measures Hypoxia-Inducible Factor 1 alpha levels in samples, aiding research on cellular responses to low oxygen levels.
What are the benefits of using the HIF-1 alpha ELISA Kit?
Using the HIF-1 alpha ELISA Kit provides a reliable method to quantify HIF-1 alpha levels. It offers a user-friendly workflow and facilitates insights into hypoxia-related pathways and disease processes.
Where can I find more information about the HIF-1 alpha ELISA Kit?
For more detailed information about the HIF-1 alpha ELISA Kit, including technical specifications, performance characteristics, and ordering details, please refer to the product brochure or contact our customer support team. We are here to assist you with any inquiries you may have.
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