Mouse Toll-like receptor 4 (Tlr4) ELISA Kit (MOEB0576)

Product Name:	Mouse Toll-like receptor 4 (Tlr4) ELISA Kit
SKU:	MOEB0576
Size:	96T
Target:	Mouse Toll-like receptor 4 (Tlr4)
Synonyms:	CD284, Lps
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Mouse
Detection Range:	0.312-20ng/mL
Sensitivity:	0.158ng/mL

Intra CV:

5.6%

Inter CV:

8.2%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	85-95%	113-113%	97-105%	92-92%
EDTA Plasma(N=5)	113-123%	83-91%	91-101%	96-105%
Heparin Plasma(N=5)	94-104%	80-90%	102-113%	84-96%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	102	96-108
Plasma	104	98-110

Function:

Cooperates with LY96 and CD14 to mediate the innate immune response to bacterial lipopolysaccharide (LPS) (PubMed:9851930, PubMed:9989976, PubMed:20133493). Acts via MYD88, TIRAP and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response (PubMed:24380872). Also involved in LPS-independent inflammatory responses triggered by free fatty acids, such as palmitate. In complex with TLR6, promotes sterile inflammation in monocytes/macrophages in response to oxidized low-density lipoprotein (oxLDL) or amyloid-beta 42. In this context, the initial signal is provided by oxLDL- or amyloid-beta 42-binding to CD36. This event induces the formation of a heterodimer of TLR4 and TLR6, which is rapidly internalized and triggers inflammatory response, leading to the NF-kappa-B-dependent production of CXCL1, CXCL2 and CCL9 cytokines, via MYD88 signaling pathway, and CCL5 cytokine, via TICAM1 signaling pathway, as well as IL1B secretion. Binds electronegative LDL (LDL(-)) and mediates the cytokine release induced by LDL(-).

Uniprot:	Q9QUK6
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant mouse Toll-like receptor 4
Sub Unit:	Belongs to the lipopolysaccharide (LPS) receptor, a multi-protein complex containing at least CD14, LY96 and TLR4 (PubMed:24380872). Binding to bacterial LPS leads to homodimerization (PubMed:20133493, PubMed:24380872, PubMed:22532668). Interacts with LY96 via the extracellular domain (PubMed:17803912, PubMed:22532668). Interacts with MYD88 (via the TIR domain). Interacts with TICAM2 and TIRAP (PubMed:24380872). Interacts with NOX4 (By similarity). Interacts with CNPY3 and HSP90B1; this interaction is required for proper folding in the endoplasmic reticulum (PubMed:18780723, PubMed:20865800). Interacts with MLK4; this interaction leads to negative regulation of TLR4 signaling (By similarity). Interacts with CD36, following CD36 stimulation by oxLDL or amyloid-beta 42, and forms a heterodimer with TLR6. The trimeric complex is internalized and triggers inflammatory response. LYN kinase activity facilitates TLR4-TLR6 heterodimerization and signal initiation (By similarity). Interacts with TICAM1 in response to LPS in a WDFY1-dependent manner (PubMed:25736436). Interacts with WDFY1 in response to LPS (PubMed:25736436). Interacts with SMPDL3B (PubMed:26095358). Interacts with CEACAM1; upon lipopolysaccharide stimulation, forms a complex including TLR4 and the phosphorylated form of SYK and CEACAM1, which in turn, recruits PTPN6 that dephosphorylates SYK, reducing the production of reactive oxygen species (ROS) and lysosome disruption, which in turn, reduces the activity of the inflammasome (PubMed:22496641).
Research Area:	Immunology
Subcellular Location:	Cell membrane Single-pass type I membrane protein Upon complex formation with CD36 and TLR6, internalized through dynamin-dependent endocytosis.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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:Receptor, misc.; Membrane protein, integral Cellular Component: cell surface; cytoplasm; external side of plasma membrane; integral to plasma membrane; intrinsic to plasma membrane; lipid raft; lipopolysaccharide receptor complex; perinuclear region of cytoplasm; plasma membrane Molecular Function:lipopolysaccharide binding; lipopolysaccharide receptor activity; phosphoinositide 3-kinase binding; protein binding; receptor activity Biological Process: activation of innate immune response; activation of MAPK activity; activation of NF-kappaB transcription factor; astrocyte development; B cell proliferation during immune response; defense response to Gram-negative bacterium; detection of lipopolysaccharide; I-kappaB phosphorylation; innate immune response; innate immune response-activating signal transduction; interferon-gamma production; interleukin-1 beta secretion; leukotriene metabolic process; lipopolysaccharide-mediated signaling pathway; macrophage activation; microglial cell activation; negative regulation of interferon-gamma production; negative regulation of interleukin-17 production; negative regulation of interleukin-23 production; negative regulation of interleukin-6 production; negative regulation of tumor necrosis factor production; positive regulation of apoptosis; positive regulation of B cell proliferation; positive regulation of chemokine production; positive regulation of DNA binding; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of inflammatory response; positive regulation of interferon-alpha production; positive regulation of interferon-beta biosynthetic process; positive regulation of interferon-beta production; positive regulation of interferon-gamma production; positive regulation of interleukin-1 production; positive regulation of interleukin-10 production; positive regulation of interleukin-12 biosynthetic process; positive regulation of interleukin-12 production; positive regulation of interleukin-6 production; positive regulation of interleukin-8 biosynthetic process; positive regulation of interleukin-8 production; positive regulation of JNK cascade; positive regulation of lymphocyte proliferation; positive regulation of MHC class II biosynthetic process; positive regulation of NF-kappaB import into nucleus; positive regulation of nitric oxide biosynthetic process; positive regulation of nitric-oxide synthase biosynthetic process; positive regulation of peptidyl-tyrosine phosphorylation; positive regulation of stress-activated MAPK cascade; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; positive regulation of tumor necrosis factor biosynthetic process; positive regulation of tumor necrosis factor production; production of nitric oxide during acute inflammatory response; regulation of dendritic cell cytokine production; regulation of inflammatory response; regulation of sensory perception of pain; response to bacterium; response to ethanol; response to lipopolysaccharide; response to oxidative stress; toll-like receptor signaling pathway
NCBI Summary:	This gene belongs to the evolutionarily-conserved Toll-like receptor family, whose members are type-1 transmembrane proteins that are involved in innate immunity. Toll-like receptors are characterized by an extracellular leucine-rich repeat domain that functions in ligand recognition and an intracellular toll/interleukin-1 receptor-like domain that is crucial for signal transduction. The receptor encoded by this gene mediates the innate immune response to bacterial lipopolysaccharide, a major component of the outer membrane of Gram-negative bacteria, through synthesis of pro-inflammatory cytokines and chemokines. In addition, this protein can recognize other pathogens from Gram-negative and Gram-positive bacteria as well as viral components. Mice deficient in this gene display a number of immune response-related phenotypes including hyporesponsiveness to bacterial lipopolysaccharide and increased levels of respiratory syncytial virus compared to controls. [provided by RefSeq, Sep 2015]
UniProt Code:	Q9QUK6
NCBI GenInfo Identifier:	20140894
NCBI Gene ID:	21898
NCBI Accession:	Q9QUK6.1
UniProt Secondary Accession:	Q9QUK6,Q9D691, Q9QZF5, Q9Z203,
UniProt Related Accession:	Q9QUK6
Molecular Weight:
NCBI Full Name:	Toll-like receptor 4
NCBI Synonym Full Names:	toll-like receptor 4
NCBI Official Symbol:	Tlr4
NCBI Official Synonym Symbols:	Lps; Ly87; Ran/M1; Rasl2-8
NCBI Protein Information:	toll-like receptor 4
UniProt Protein Name:	Toll-like receptor 4
UniProt Synonym Protein Names:	CD_antigen: CD284
Protein Family:	Toll-like receptor
UniProt Gene Name:	Tlr4
UniProt Entry Name:	TLR4_MOUSE

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.

Mouse Toll-like receptor 4 (Tlr4) ELISA Kit (MOEB0576)

Description