Human Toll-like receptor 4 (TLR4) ELISA Kit

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SKU:
HUEB0840
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
O00206
Range:
0.625-40 ng/mL
ELISA Type:
Sandwich
Synonyms:
TLR4, CD284, ARMD10, CD284 antigen, hTollhomolog of Drosophila toll, TOLL, toll-like receptor 4
Reactivity:
Human
Frequently bought together:

Description

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Product Name:Human Toll-like receptor 4 (TLR4) ELISA Kit
Product Code:HUEB0840
Alias:Toll-like receptor 4, hToll, TLR4, CD284
Uniprot:O00206
Reactivity:Human
Range:0.625-40 ng/mL
Detection Method:Sandwich
Size:96 Assay
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: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
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.
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