The Mouse Toll-like Receptor 3 (TLR3) ELISA Kit is specially designed for the precise measurement of TLR3 levels in mouse serum, plasma, and tissue homogenates. With its exceptional sensitivity and specificity, this kit ensures accurate and reproducible results, making it an ideal tool for various research applications.TLR3 is a key component of the immune system's response to viral infections, triggering the production of inflammatory cytokines and interferons.
Dysregulation of TLR3 signaling has been linked to autoimmune diseases, viral infections, and inflammatory conditions, highlighting the importance of studying TLR3 levels in these pathological states.By using the Mouse TLR3 ELISA Kit, researchers can gain valuable insights into the role of TLR3 in immune responses and disease pathogenesis, paving the way for the development of novel therapeutic interventions targeting TLR3 signaling pathways.
Product Name:
Mouse Toll-like receptor 3 (Tlr3) ELISA Kit
SKU:
MOEB1836
Size:
96T
Target:
Mouse Toll-like receptor 3 (Tlr3)
Synonyms:
CD283
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Mouse
Detection Range:
78-5000pg/mL
Sensitivity:
30pg/mL
Intra CV:
Provided with the Kit
Inter CV:
Provided with the Kit
Linearity:
Provided with the Kit
Recovery:
Provided with the Kit
Function:
Key component of innate and adaptive immunity. TLRs (Toll-like receptors) control host immune response against pathogens through recognition of molecular patterns specific to microorganisms. TLR3 is a nucleotide-sensing TLR which is activated by double-stranded RNA, a sign of viral infection. Acts via the adapter TRIF/TICAM1, leading to NF-kappa-B activation, IRF3 nuclear translocation, cytokine secretion and the inflammatory response.
Uniprot:
Q99MB1
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse Toll-like receptor 3
Sub Unit:
Monomer and homodimer; dimerization is triggered by ligand-binding, the signaling unit is composed of one ds-RNA of around 40 bp and two TLR3 molecules, and lateral clustering of signaling units along the length of the ds-RNA ligand is required for TLR3 signal transduction. Interacts (via transmembrane domain) with UNC93B1; the interaction is required for transport from the ER to the endosomes. Interacts with SRC; upon binding of double-stranded RNA (By similarity). Interacts with TICAM1 (via the TIR domain) in response to poly(I:C) and this interaction is enhanced in the presence of WDFY1 (PubMed:25736436). The tyrosine-phosphorylated form (via TIR domain) interacts with WDFY1 (via WD repeat 2) in response to poly(I:C) (PubMed:25736436).
Research Area:
Immunology
Subcellular Location:
Endoplasmic reticulum membrane Single-pass type I membrane protein Endosome membrane Early endosome
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
TLR3: a member of the Toll-like receptor (TLR) family which plays a fundamental role in pathogen recognition and activation of innate immunity. Most abundantly expressed in placenta and pancreas. Restricted to the dendritic subpopulation of the leukocytes. It recognizes dsRNA associated with viral infection. Acts via MyD88 and TRAF6, leading to the activation of NF-kappaB and the production of type I interferon. Protein type: Receptor, cytokine; Membrane protein, integralCellular Component: cell surface; membrane; lysosomal membrane; endoplasmic reticulum; cytoplasm; integral to membrane; intracellular; endosomeMolecular Function: identical protein binding; protein binding; transmembrane receptor activity; RNA binding; double-stranded RNA binding; receptor activityBiological Process: positive regulation of interleukin-12 production; microglial cell activation; positive regulation of JNK cascade; positive regulation of NF-kappaB import into nucleus; positive regulation of interferon-alpha biosynthetic process; defense response; toll-like receptor 3 signaling pathway; signal transduction; activation of NF-kappaB transcription factor; positive regulation of interleukin-8 production; response to exogenous dsRNA; positive regulation of interferon-beta production; positive regulation of interferon type I production; inflammatory response; defense response to virus; positive regulation of cytokine production; positive regulation of I-kappaB kinase/NF-kappaB cascade; immune system process; response to virus; MyD88-independent toll-like receptor signaling pathway; response to dsRNA; positive regulation of interleukin-6 production; positive regulation of tumor necrosis factor production; positive regulation of chemokine production; positive regulation of toll-like receptor signaling pathway; positive regulation of chemokine biosynthetic process; positive regulation of interferon-beta biosynthetic process; toll-like receptor signaling pathway; innate immune response; positive regulation of transcription from RNA polymerase II promoter; positive regulation of protein amino acid phosphorylation; I-kappaB phosphorylation; positive regulation of interferon-gamma biosynthetic process; microglial cell activation during immune response; positive regulation of inflammatory response
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.
