Description
Human IRF3 ELISA Kit
The Human IRF3 (Interferon Regulatory Factor 3) ELISA Kit is a reliable and accurate tool for the quantitative measurement of IRF3 levels in human samples, including serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit delivers consistent and reproducible results, making it an ideal choice for various research applications.IRF3 is a key transcription factor involved in the regulation of interferon production and immune response to viral infections. Dysregulation of IRF3 signaling has been implicated in autoimmune diseases, viral infections, and inflammatory disorders, highlighting its importance as a potential therapeutic target and biomarker for disease progression.
By utilizing the Human IRF3 ELISA Kit, researchers can gain valuable insights into the role of IRF3 in disease pathogenesis and explore its potential as a target for novel treatment strategies. This kit offers a convenient and efficient solution for studying IRF3 biology and its implications in various disease contexts.
| Product Name: | Human IRF3 ELISA Kit |
| Product Code: | HUFI01380 |
| Size: | 96 Assays |
| Alias: | IRF3, Interferon regulatory factor 3, IRF-3 |
| Detection method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human IRF3 concentrations in serum plasma and other biological fluids. |
| Sensitivity: | 0.188ng/ml |
| Range: | 0.313-20ng/ml |
| Storage: | 4°C for 6 months |
| Note: | For Research Use Only |
| Recovery: | Matrices listed below were spiked with certain level of Human IRF3 and the recovery rates were calculated by comparing the measured value to the expected amount of Human IRF3 in samples. | ||||||||||||||||
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| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human IRF3 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% Inter-Assay: CV<10% |
| Component | Quantity | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | 4°C for 6 months |
| Lyophilized Standard | 2 | 4°C/-20°C |
| Sample/Standard Dilution Buffer | 20ml | 4°C |
| Biotin-labeled Antibody(Concentrated) | 120ul | 4°C (Protect 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 (Protect from light) |
| Stop Solution | 10ml | 4°C |
| Wash Buffer(25X) | 30ml | 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
| Uniprot | Q14653 |
| UniProt Protein Function: | IRF3: interferon regulatory factor 3, a member of the interferon regulatory transcription factor (IRF) family. IRF3 is found in an inactive cytoplasmic form that upon serine/threonine phosphorylation forms a complex with CREBBP. This complex translocates to the nucleus and activates the transcription of interferons alpha and beta, as well as other interferon-induced genes. |
| UniProt Protein Details: | Protein type:DNA-binding; Transcription factor Chromosomal Location of Human Ortholog: 19q13.3-q13.4 Cellular Component: nucleoplasm; cytoplasm; nucleus; cytosol Molecular Function:identical protein binding; protein binding; protein homodimerization activity; DNA binding; transcription cofactor activity; transcription factor activity Biological Process: transcription from RNA polymerase II promoter; negative regulation of interferon-beta biosynthetic process; positive regulation of I-kappaB kinase/NF-kappaB cascade; viral reproduction; apoptosis; MyD88-independent toll-like receptor signaling pathway; cytokine and chemokine mediated signaling pathway; toll-like receptor 3 signaling pathway; negative regulation of transcription from RNA polymerase II promoter; negative regulation of defense response to virus by host; response to exogenous dsRNA; positive regulation of interferon type I production; lipopolysaccharide-mediated signaling pathway; toll-like receptor signaling pathway; positive regulation of interferon-beta production; innate immune response; interferon type I biosynthetic process; defense response to virus; toll-like receptor 4 signaling pathway; response to DNA damage stimulus; negative regulation of interferon type I production; positive regulation of interferon-alpha production; positive regulation of cytokine secretion Disease: Herpes Simplex Encephalitis, Susceptibility To, 7 |
| NCBI Summary: | This gene encodes a member of the interferon regulatory transcription factor (IRF) family. The encoded protein is found in an inactive cytoplasmic form that upon serine/threonine phosphorylation forms a complex with CREBBP. This complex translocates to the nucleus and activates the transcription of interferons alpha and beta, as well as other interferon-induced genes. Alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene. [provided by RefSeq, Nov 2011] |
| UniProt Code: | Q14653 |
| NCBI GenInfo Identifier: | 2497442 |
| NCBI Gene ID: | 3661 |
| NCBI Accession: | Q14653.1 |
| UniProt Secondary Accession: | Q14653,Q5FBY1, Q5FBY2, Q5FBY4, Q7Z5G6, A8K7L2, B2RAZ3 |
| UniProt Related Accession: | Q14653 |
| Molecular Weight: | 12,330 Da |
| NCBI Full Name: | Interferon regulatory factor 3 |
| NCBI Synonym Full Names: | interferon regulatory factor 3 |
| NCBI Official Symbol: | IRF3 |
| NCBI Protein Information: | interferon regulatory factor 3 |
| UniProt Protein Name: | Interferon regulatory factor 3 |
| Protein Family: | Interferon regulatory factor |
| UniProt Gene Name: | IRF3 |
| UniProt Entry Name: | IRF3_HUMAN |
*Note: Protocols are specific to each batch/lot. For the correct 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 | Protocol |
| 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. |
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. |
