Human TLR4 ELISA Kit
- SKU:
- HUFI00788
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- O00206
- Sensitivity:
- 0.188ng/ml
- Range:
- 0.313-20ng/ml
- ELISA Type:
- Sandwich
- Synonyms:
- TLR4, CD284, ARMD10, CD284 antigen, hTollhomolog of Drosophila toll, TOLL, toll-like receptor 4
- Reactivity:
- Human
- Research Area:
- Immunology
Description
Human TLR4 ELISA Kit
The TLR4 receptor recognizes LPS from Gram-negative bacteria and other bacterial cell wall components. This binding activates the receptor and triggers downstream signaling pathways, leading to inflammatory responses which include increased cytokine production, increased expression of adhesion molecules on endothelial cells, and activation of leukocytes such as neutrophils and macrophages. As such, TLR4 plays a key role in the immune system and is used as a marker for certain autoimmune diseases, such as Crohn's disease.
| Product Name: | Human TLR4 ELISA Kit |
| Product Code: | HUFI00788 |
| Size: | 96 Assays |
| Alias: | TLR4, CD284, ARMD10, CD284 antigen, hTollhomolog of Drosophila toll, TOLL, toll-like receptor 4 |
| Detection method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human TLR4 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 TLR4 and the recovery rates were calculated by comparing the measured value to the expected amount of Human TLR4 in samples. | ||||||||||||||||
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| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human TLR4 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 | O00206 |
| UniProt Protein Function: | TLR4: Cooperates with LY96 and CD14 to mediate the innate immune response to bacterial lipopolysaccharide (LPS). Acts via MYD88, TIRAP and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response. Also involved in LPS- independent inflammatory responses triggered by Ni(2+). These responses require non-conserved histidines and are, therefore, species-specific. Belongs to the lipopolysaccharide (LPS) receptor, a multi-protein complex containing at least CD14, LY96 and TLR4. Binding to bacterial LPS leads to homodimerization. Interacts with LY96 via the extracellular domain. Interacts with MYD88 and TIRAP via their respective TIR domains. Interacts with NOX4. Interacts with CNPY3. Interacts with HSP90B1. The interaction with both CNPY3 and HSP90B1 is required for proper folding in the endoplasmic reticulum. Highly expressed in placenta, spleen and peripheral blood leukocytes. Detected in monocytes, macrophages, dendritic cells and several types of T-cells. Belongs to the Toll-like receptor family. 3 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Membrane protein, integral; Receptor, misc. Chromosomal Location of Human Ortholog: 9q33.1 Cellular Component: integral to plasma membrane; perinuclear region of cytoplasm; cytoplasm; plasma membrane; lipopolysaccharide receptor complex; endosome membrane; external side of plasma membrane Molecular Function:protein binding; transmembrane receptor activity; lipopolysaccharide binding; lipopolysaccharide receptor activity; receptor activity Biological Process: I-kappaB kinase/NF-kappaB cascade; positive regulation of nitric oxide biosynthetic process; activation of MAPK activity; response to lipopolysaccharide; positive regulation of NF-kappaB import into nucleus; toll-like receptor 3 signaling pathway; positive regulation of interleukin-10 production; activation of NF-kappaB transcription factor; positive regulation of interferon-beta production; negative regulation of interleukin-6 production; positive regulation of B cell proliferation; toll-like receptor 4 signaling pathway; positive regulation of interferon-alpha production; T-helper 1 type immune response; production of nitric oxide during acute inflammatory response; positive regulation of interleukin-6 production; positive regulation of interleukin-8 biosynthetic process; positive regulation of tumor necrosis factor production; positive regulation of chemokine production; toll-like receptor 2 signaling pathway; negative regulation of interleukin-23 production; macrophage activation; detection of lipopolysaccharide; defense response to bacterium; positive regulation of transcription from RNA polymerase II promoter; I-kappaB phosphorylation; positive regulation of nitric-oxide synthase biosynthetic process; negative regulation of osteoclast differentiation; regulation of cytokine secretion; positive regulation of interleukin-12 production; positive regulation of JNK cascade; defense response to Gram-negative bacterium; positive regulation of interleukin-8 production; negative regulation of interferon-gamma production; positive regulation of MHC class II biosynthetic process; interferon-gamma production; lipopolysaccharide-mediated signaling pathway; B cell proliferation during immune response; positive regulation of tumor necrosis factor biosynthetic process; detection of fungus; MyD88-independent toll-like receptor signaling pathway; negative regulation of tumor necrosis factor production; MyD88-dependent toll-like receptor signaling pathway; positive regulation of interferon-gamma production; positive regulation of interleukin-12 biosynthetic process; negative regulation of interleukin-17 production; positive regulation of interferon-beta biosynthetic process; toll-like receptor signaling pathway; innate immune response; immune response; positive regulation of interleukin-1 production; positive regulation of inflammatory response Disease: Macular Degeneration, Age-related, 10 |
| NCBI Summary: | The protein encoded by this gene is a member of the Toll-like receptor (TLR) family which plays a fundamental role in pathogen recognition and activation of innate immunity. TLRs are highly conserved from Drosophila to humans and share structural and functional similarities. They recognize pathogen-associated molecular patterns that are expressed on infectious agents, and mediate the production of cytokines necessary for the development of effective immunity. The various TLRs exhibit different patterns of expression. This receptor has been implicated in signal transduction events induced by lipopolysaccharide (LPS) found in most gram-negative bacteria. Mutations in this gene have been associated with differences in LPS responsiveness. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jan 2012] |
| UniProt Code: | O00206 |
| NCBI GenInfo Identifier: | 20140413 |
| NCBI Gene ID: | 7099 |
| NCBI Accession: | O00206.2 |
| UniProt Secondary Accession: | O00206,Q5VZI8, Q5VZI9, Q9UK78, Q9UM57, A8K1Y8, A9XLP9 A9XLQ0, A9XLQ1, B4E194, D1CS52, D1CS53, |
| UniProt Related Accession: | O00206 |
| Molecular Weight: | 73,301 Da |
| NCBI Full Name: | Toll-like receptor 4 |
| NCBI Synonym Full Names: | toll-like receptor 4 |
| NCBI Official Symbol: | TLR4 |
| NCBI Official Synonym Symbols: | TOLL; CD284; TLR-4; ARMD10 |
| NCBI Protein Information: | toll-like receptor 4; hToll; homolog of Drosophila toll |
| UniProt Protein Name: | Toll-like receptor 4 |
| UniProt Synonym Protein Names: | hToll; CD_antigen: CD284 |
| Protein Family: | Toll-like receptor |
| UniProt Gene Name: | TLR4 |
| UniProt Entry Name: | TLR4_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. |
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