Human TLR4 Antibody

(13 citations)   
  • Species Reactivity
    Human
  • Specificity
    Detects human TLR4 in direct ELISAs and Western blots. In direct ELISAs and Western blots, approximately 30% cross-reactivity with recombinant mouse (rm) TLR4 is observed and less than 5% cross-reactivity with recombinant human (rh) TLR1, rhTLR2, rhTLR3 and rmTLR6 is observed.
  • Source
    Polyclonal Goat IgG
  • Purification
    Antigen Affinity-purified
  • Immunogen
    Mouse myeloma cell line NS0-derived recombinant human TLR4
    Glu24-Lys631
    Accession # O00206
  • Formulation
    Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied as a 0.2 µm filtered solution in PBS.
  • Endotoxin Level
    <0.10 EU per 1 μg of the antibody by the LAL method.
  • Label
    Unconjugated
Applications
  •  
    Recommended
    Concentration
    Sample
  • Western Blot
    1 µg/mL
    See below
  • Flow Cytometry
    2.5 µg/106 cells
    Human peripheral blood monocytes
  • Immunohistochemistry
    5-15 µg/mL
    See below
  • CyTOF-ready
    Ready to be labeled using established conjugation methods. No BSA or other carrier proteins that could interfere with conjugation.
  • Immunocytochemistry
    5-15 µg/mL
    See below
  • Neutralization
    Measured by its ability to neutralize LPS-induced IL‑8 secretion in the HEK293 human embryonic kidney cell line co-transfected with human TLR4 and MD-2. The Neutralization Dose (ND50) is typically 1.5-7.5 µg/mL in the presence of 75 ng/mL Lipopolysacharide (LPS).
Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website.
Data Examples
Detection of Human TLR4 by Western Blot. Western blot shows lysates of human placenta tissue. PVDF membrane was probed with 1 µg/mL of Goat Anti-Human TLR4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1478) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). Specific bands were detected for TLR4 at approximately 110 kDa and 130 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 8.
Immunocytochemistry
TLR4 in Human PBMCs. TLR4 was detected in immersion fixed human peripheral blood mononuclear cells (PBMCs) using Goat Anti-Human TLR4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1478) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (yellow; Catalog # NL001) and counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Non-adherent Cells.
Immunohistochemistry
TLR4 in Human Prostate. TLR4 was detected in immersion fixed paraffin-embedded sections of human prostate using Goat Anti-Human TLR4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1478) at 10 µg/mL overnight at 4 °C. Before incubation with the primary antibody tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.
IL‑8 Secretion Induced by LPS and Neutralization by Human TLR4 Antibody. Lipopolysacharide (LPS) stimulates IL‑8 secretion in the HEK293 human embryonic kidney cell line co-transfected with human TLR4 and MD-2, in a dose-dependent manner (orange line), as measured by the Human CXCL8/IL‑8 Quantikine ELISA Kit (Catalog # D8000C). IL‑8 secretion elicited by LPS (75 ng/mL) is neutralized (green line) by increasing concentrations of Goat Anti-Human TLR4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1478). The ND50 is typically 1.5-7.5 µg/mL.
Preparation and Storage
  • Reconstitution
    Reconstitute at 0.2 mg/mL in sterile PBS.
  • Shipping
    The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. *Small pack size (SP) is shipped with polar packs. Upon receipt, store it immediately at -20 to -70 °C
  • Stability & Storage
    Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
    • 12 months from date of receipt, -20 to -70 °C as supplied.
    • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
    • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
Background: TLR4

TLR4 is a 100 kDa type I transmembrane glycoprotein that belongs to the mammalian Toll-Like Receptor family of pathogen pattern recognition molecules. In the literature molecular weights correspondent to 110 kDa and 130 kDa were reported for TLR4 (1). MD-2, also known as ESOP-1, is a 25 kDa secreted protein that is required for TLR4-mediated responses to bacterial lipopolysaccharide (LPS) (1‑4). The human TLR4 cDNA encodes an 839 amino acid (aa) precursor that contains a 23 aa signal sequence, a 608 aa extracellular domain (ECD), a 21 aa transmembrane segment, and a 187 aa cytoplasmic domain. TLR4 contains 21 leucine rich repeats in its ECD and one cytoplasmic Toll/IL-1 receptor (TIR) domain (5). The ECD of human TLR4 shares approximately 25% aa sequence identity with other TLRs and 60%‑74% aa sequence identity with bovine, equine, feline, mouse, rat, and porcine TLR4. The human MD-2 cDNA encodes a 160 aa precursor with an 18 aa signal sequence (5). Human MD-2 shares 20% aa sequence identity with MD-1 and 62%‑64% aa sequence identity with bovine, mouse, and rat MD-2. MD-2 associates with TLR4 on monocytes, macrophages, dendritic cells, and B cells (6‑8). MD-2 expression is required for cell surface localization of TLR4 and for optimal LPS-induced TLR4 signaling (8, 9). MD-2 also forms soluble disulfide-linked homo-oligomers which can interact with TLR4 (7). Through a domain separate from its TLR4-binding domain, MD-2 extracts LPS from circulating CD14-LPS complexes and carries the LPS into a ternary complex with TLR4 (10‑12). The interaction of MD-2/LPS with TLR4 induces receptor oligomerization and the triggering of an inflammatory response (13). Increased levels of plasma MD-2 in septic shock patients sensitizes MD-2 non-expressing epithelial cells to LPS and promotes widespread tissue inflammation (14).

  • References:
    1. Ohnishi, T. et al. (2003) Clin. Diagn. Lab. Immouol. 10:405.
    2. Gangloff, M. and N.J. Gay (2004) Trends Biochem. Sci. 29:294.
    3. Palsson-McDermott, E.M. and L.A. O’Neill (2004) Immunology 113:153.
    4. Miyake, K. (2004) Semin. Immunol. 16:11.
    5. Medzhitov, R. et al. (1997) Nature 388:394.
    6. Shimazu, R. et al. (1999) J. Exp. Med. 189:1777.
    7. Visintin, A. et al. (2001) Proc. Natl. Acad. Sci. USA 98:12156.
    8. Akashi, S. et al. (2000) J. Immunol. 164:3471.
    9. Nagai, Y. et al. (2002) Nat. Immunol. 3:667.
    10. Re, F. and J.L. Strominger (2003) J. Immunol. 171:5272.
    11. Kennedy, M.N. et al. (2004) J. Biol. Chem. 279:34698.
    12. Gioannini, T.L. et al. (2004) Proc. Natl. Acad. Sci. USA 101:4186.
    13. Saitoh, S. et al. (2004) J. Endotoxin Res. 10:257.
    14. Pugin, J. et al. (2004) Blood 104:4071.
  • Long Name:
    Toll-like Receptor 4
  • Entrez Gene IDs:
    7099 (Human); 21898 (Mouse); 29260 (Rat)
  • Alternate Names:
    ARMD10; CD284 antigen; CD284; hTollhomolog of Drosophila toll; TLR4; TOLL; toll-like receptor 4
Related Research Areas
Citations:

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

13 Citations: Showing 1 - 10
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Species
Applications
Sample Type
  1. TLR4 Expression Is Associated with Left Ventricular Dysfunction in Patients Undergoing Coronary Artery Bypass Surgery.
    Authors: Avlas O, Bragg A, Fuks A, Nicholson J, Farkash A, Porat E, Aravot D, Levy-Drummer R, Cohen C, Shainberg A, Arad M, Hochhauser E
    PLoS ONE, 2015;10(6):e0120175.
    Species: Human
    Sample Type: Whole Tissue
    Application: IHC Frozen
  2. The C-terminal disulfide bonds of Helicobacter pylori GroES are critical for IL-8 secretion via the TLR4-dependent pathway in gastric epithelial cells.
    Authors: Su Y, Yang J, Lee H, Sheu F, Hsu C, Lin S, Chow L
    J Immunol, 2015;194(8):3997-4007.
    Species: Helicobacter pylori
    Sample Type: Cell Lysates
    Application: IP
  3. HIV-1 Tat Protein Induces Production of Proinflammatory Cytokines by Human Dendritic Cells and Monocytes/Macrophages through Engagement of TLR4-MD2-CD14 Complex and Activation of NF-kappaB Pathway.
    Authors: Ben Haij N, Planes R, Leghmari K, Serrero M, Delobel P, Izopet J, BenMohamed L, Bahraoui E
    PLoS ONE, 2015;10(6):e0129425.
    Species: Human
    Sample Type: Whole Cells
    Application: Flow
  4. Necrotic cell-derived high mobility group box 1 attracts antigen-presenting cells but inhibits hepatocyte growth factor-mediated tropism of mesenchymal stem cells for apoptotic cell death.
    Authors: Vogel S, Borger V, Peters C, Forster M, Liebfried P, Metzger K, Meisel R, Daubener W, Trapp T, Fischer J, Gawaz M, Sorg R
    Cell Death Differ, 2015;22(7):1219-30.
    Species: Mouse
    Sample Type: Whole Cells
    Application: Neut
  5. Slit2-Robo4 pathway modulates lipopolysaccharide-induced endothelial inflammation and its expression is dysregulated during endotoxemia.
    Authors: Zhao H, Anand A, Ganju R
    J Immunol, 2014;192(1):385-93.
    Species: Human
    Sample Type: Whole Cells
    Application: Neut
  6. Pathogenic strains of Acanthamoeba are recognized by TLR4 and initiated inflammatory responses in the cornea.
    Authors: Alizadeh H, Tripathi T, Abdi M, Smith A
    PLoS ONE, 2014;9(3):e92375.
    Species: Human
    Sample Type: Whole Cells
    Application: Neut
  7. Serum amyloid A in the placenta and its role in trophoblast invasion.
    Authors: Sandri S, Urban Borbely A, Fernandes I, de Oliveira E, Knebel F, Ruano R, Zugaib M, Filippin-Monteiro F, Bevilacqua E, Campa A
    PLoS ONE, 2014;9(3):e90881.
    Species: Human
    Sample Type: Whole Cells
    Application: Functional Assay
  8. Effect of thrombin on human amnion mesenchymal cells, mouse fetal membranes, and preterm birth.
    Authors: Mogami, Haruta, Keller, Patrick, Shi, Haolin, Word, R Ann
    J Biol Chem, 2014;289(19):13295-307.
    Species: Human
    Sample Type: Whole Cells
    Application: Neut
  9. Activated platelets interfere with recruitment of mesenchymal stem cells to apoptotic cardiac cells via high mobility group box 1/Toll-like receptor 4-mediated down-regulation of hepatocyte growth factor receptor MET.
    Authors: Vogel S, Chatterjee M, Metzger K, Borst O, Geisler T, Seizer P, Muller I, Mack A, Schumann S, Buhring H, Lang F, Sorg R, Langer H, Gawaz M
    J Biol Chem, 2014;289(16):11068-82.
    Species: Human
    Sample Type: Whole Cells
    Application: Flow
  10. Regulation of the innate immune response by fibronectin: synergism between the III-1 and EDA domains.
    Authors: Kelsh R, You R, Horzempa C, Zheng M, McKeown-Longo P
    PLoS ONE, 2014;9(7):e102974.
    Species: Human
    Sample Type: Whole Cells
    Application: Neut
  11. Fetal fibronectin signaling induces matrix metalloproteases and cyclooxygenase-2 (COX-2) in amnion cells and preterm birth in mice.
    Authors: Mogami H, Kishore A, Shi H, Keller P, Akgul Y, Word R
    J Biol Chem, 2013;288(3):1953-66.
    Species: Human
    Sample Type: Whole Cells
    Application: Neut
  12. Isotretinoin increases skin-surface levels of neutrophil gelatinase-associated lipocalin in patients treated for severe acne.
    Authors: Lumsden KR, Nelson AM, Dispenza MC, Gilliland KL, Cong Z, Zaenglein AL, Thiboutot DM
    Br. J. Dermatol., 2011;165(2):302-10.
    Species: Human
    Sample Type: Whole Cells
    Application: Neut
  13. Soluble forms of Toll-like receptor 4 are present in human saliva and modulate tumour necrosis factor-alpha secretion by macrophage-like cells.
    Authors: Zunt SL, Burton LV, Goldblatt LI, Dobbins EE, Srinivasan M
    Clin. Exp. Immunol., 2009;156(2):285-93.
    Species: Human
    Sample Type: Saliva
    Application: WB
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