Human Toll-like receptor 2 (TLR2) ELISA Kit

Product Name:	Human Toll-like receptor 2 (TLR2) ELISA Kit
SKU:	HUEB0788
Size:	96T
Target:	Human Toll-like receptor 2 (TLR2)
Synonyms:	Toll/interleukin-1 receptor-like protein 4, CD282, TIL4
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.312-20ng/mL
Sensitivity:	0.172ng/mL

Intra CV:

4.1%

Inter CV:

7.7%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	100-110%	97-107%	107-117%	102-112%
EDTA Plasma(N=5)	99-110%	94-105%	107-117%	103-113%
Heparin Plasma(N=5)	102-111%	105-117%	102-110%	95-108%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	97	91-103
Plasma	99	93-105

Function:

Cooperates with LY96 to mediate the innate immune response to bacterial lipoproteins and other microbial cell wall components. Cooperates with TLR1 or TLR6 to mediate the innate immune response to bacterial lipoproteins or lipopeptides (PubMed:21078852, PubMed:17889651). Acts via MYD88 and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response. May also activate immune cells and promote apoptosis in response to the lipid moiety of lipoproteins (PubMed:10426995, PubMed:10426996). Recognizes mycoplasmal macrophage-activating lipopeptide-2kD (MALP-2), soluble tuberculosis factor (STF), phenol-soluble modulin (PSM) and B.burgdorferi outer surface protein A lipoprotein (OspA-L) cooperatively with TLR6 (PubMed:11441107). Stimulation of monocytes in vitro with M.tuberculosis PstS1 induces p38 MAPK and ERK1/2 activation primarily via this receptor, but also partially via TLR4 (PubMed:16622205). MAPK activation in response to bacterial peptidoglycan also occurs via this receptor (PubMed:16622205). Acts as a receptor for M.tuberculosis lipoproteins LprA, LprG, LpqH and PstS1, some lipoproteins are dependent on other coreceptors (TLR1, CD14 and/or CD36); the lipoproteins act as agonists to modulate antigen presenting cell functions in response to the pathogen (PubMed:19362712). M.tuberculosis HSP70 (dnaK) but not HSP65 (groEL-2) acts via this protein to stimulate NF-kappa-B expression (PubMed:15809303). Recognizes M.tuberculosis major T-antigen EsxA (ESAT-6) which inhibits downstream MYD88-dependent signaling (shown in mouse) (By similarity). Forms activation clusters composed of several receptors depending on the ligand, these clusters trigger signaling from the cell surface and subsequently are targeted to the Golgi in a lipid-raft dependent pathway. Forms the cluster TLR2:TLR6:CD14:CD36 in response to diacylated lipopeptides and TLR2:TLR1:CD14 in response to triacylated lipopeptides (PubMed:16880211). Required for normal uptake of M.tuberculosis, a process that is inhibited by M.tuberculosis LppM.

Uniprot:	O60603
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Toll-like receptor 2
Sub Unit:	(Microbial infection) Interacts with M.tuberculosis EsxA (PubMed:20800577). Interacts with M.bovis MPB83 (PubMed:20800577).
Research Area:	Immunology
Subcellular Location:	Membrane Single-pass type I membrane protein Cytoplasmic vesicle Phagosome membrane Single-pass type I membrane protein Membrane raft Does not reside in lipid rafts before stimulation but accumulates increasingly in the raft upon the presence of the microbial ligand. In response to diacylated lipoproteins, TLR2:TLR6 heterodimers are recruited in lipid rafts, this recruitment determines the intracellular targeting to the Golgi apparatus. Triacylated lipoproteins induce the same mechanism for TLR2:TLR1 heterodimers.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	TLR2: Cooperates with LY96 to mediate the innate immune response to bacterial lipoproteins and other microbial cell wall components. Cooperates with TLR1 to mediate the innate immune response to bacterial lipoproteins or lipopeptides. Acts via MYD88 and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response. May also promote apoptosis in response to lipoproteins. Recognizes mycoplasmal macrophage- activating lipopeptide-2kD (MALP-2), soluble tuberculosis factor (STF), phenol-soluble modulin (PSM) and B.burgdorferi outer surface protein A lipoprotein (OspA-L) cooperatively with TLR6. Interacts with LY96, TLR1 and TLR6 (via extracellular domain). Binds MYD88 (via TIR domain). Interacts with TICAM1. Ligand binding induces the formation of a heterodimer with TLR1. Interacts with CNPY3. Highly expressed in peripheral blood leukocytes, in particular in monocytes, in bone marrow, lymph node and in spleen. Also detected in lung and in fetal liver. Levels are low in other tissues. Belongs to the Toll-like receptor family.
UniProt Protein Details:	Protein type:Membrane protein, integral; Apoptosis; Motility/polarity/chemotaxis; Cell surface; Receptor, misc. Chromosomal Location of Human Ortholog: 4q32 Cellular Component: cell surface; cell projection; integral to plasma membrane; cytoplasm; plasma membrane; external side of plasma membrane Molecular Function:peptidoglycan binding; protein binding; transmembrane receptor activity; triacylated lipoprotein binding; protein heterodimerization activity; lipopolysaccharide receptor activity; receptor activity; pattern recognition receptor activity; diacylated lipoprotein binding Biological Process: positive regulation of nitric oxide biosynthetic process; apoptosis; positive regulation of interleukin-12 production; response to toxin; positive regulation of leukocyte migration; microglial cell activation; leukotriene metabolic process; positive regulation of NF-kappaB import into nucleus; response to molecule of fungal origin; detection of triacylated bacterial lipoprotein; positive regulation of interleukin-18 production; signal transduction; nitric oxide metabolic process; positive regulation of interleukin-10 production; response to insulin stimulus; activation of NF-kappaB transcription factor; positive regulation of interleukin-8 production; negative regulation of cell proliferation; lipopolysaccharide-mediated signaling pathway; positive regulation of interferon-beta production; positive regulation of oligodendrocyte differentiation; inflammatory response; toll-like receptor 4 signaling pathway; positive regulation of Wnt receptor signaling pathway; positive regulation of tumor necrosis factor biosynthetic process; negative regulation of interleukin-12 production; detection of diacylated bacterial lipoprotein; cell surface pattern recognition receptor signaling pathway; positive regulation of interleukin-6 production; positive regulation of tumor necrosis factor production; positive regulation of toll-like receptor signaling pathway; positive regulation of chemokine production; toll-like receptor 2 signaling pathway; MyD88-dependent toll-like receptor signaling pathway; induction by symbiont of defense-related host nitric oxide production; defense response to Gram-positive bacterium; negative regulation of interleukin-17 production; myelin formation in the central nervous system; response to hypoxia; toll-like receptor signaling pathway; innate immune response; positive regulation of transcription from RNA polymerase II promoter; immune response; response to progesterone stimulus; I-kappaB phosphorylation; chloramphenicol transport; positive regulation of cytokine secretion; positive regulation of nitric-oxide synthase biosynthetic process; positive regulation of inflammatory response Disease: Leprosy, Susceptibility To, 3; Mycobacterium Tuberculosis, Susceptibility To
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 (PAMPs) 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 gene is expressed most abundantly in peripheral blood leukocytes, and mediates host response to Gram-positive bacteria and yeast via stimulation of NF-kappaB. [provided by RefSeq, Jul 2008]
UniProt Code:	O60603
NCBI GenInfo Identifier:	20140434
NCBI Gene ID:	7097
NCBI Accession:	O60603.1
UniProt Secondary Accession:	O60603,O15454, Q8NI00, B3Y612, D1CS45, D1CS48, D1CS49
UniProt Related Accession:	O60603
Molecular Weight:	89,838 Da
NCBI Full Name:	Toll-like receptor 2
NCBI Synonym Full Names:	toll-like receptor 2
NCBI Official Symbol:	TLR2
NCBI Official Synonym Symbols:	TIL4; CD282
NCBI Protein Information:	toll-like receptor 2
UniProt Protein Name:	Toll-like receptor 2
UniProt Synonym Protein Names:	Toll/interleukin-1 receptor-like protein 4; CD_antigen: CD282
Protein Family:	Toll-like receptor
UniProt Gene Name:	TLR2
UniProt Entry Name:	TLR2_HUMAN

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.

ELISA	Antibodies
Human TLR2 (Toll-Like Receptor 2) ELISA Kit (HUES02074)	Anti-TLR2 Antibody (CAB0367)
	Anti-TLR2 Antibody (CAB11225)
	Anti-TLR2 Antibody (CAB19125)
	Anti-TLR2 Antibody (CAB2545)
	TLR2 Antibody (PACO03218)