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Datasheet
Monoclonal Antibody to human TLR4 (Toll-like receptor 4)/CD284
Cat.No IMG-417A
Description TLR4/CD284
Format Purified
Unit 0.1 mg
Price(USD) 255.00
MSDS
Catalog No
:
IMG-417A
Contents
:
100 µg in 200 µl PBS containing 0.05% BSA and 0.05% sodium azide. Sodium azide is highly toxic.
Isotype
:
Mouse IgG2a, Kappa
Clone
:
HTA125
Purification
:
Protein G Chromatography
Species
:
Dog, Human
Host
:
Mouse
Application
Confocal Microscopy: see Scheel et al. (2006) for details FA(Neutralization): see Paik et al, 2003 and Basek et al, 2005 for details. Flow (Cell Surface): 2-3 ug/10^6 cells Flow (Intracellular): see Wong et al. (2007) for details IF/ICC: see Schneeman et al (2005) for details. IP: see Shimazu et al, 1999 for details Storage Store at 4°C, stable for 6 months. For long-term storage, store at -20°C. Recommended Positive Control : PBMCs or ThP1
Background The Toll-like receptor (TLR) family in mammal comprises a family of transmembrane proteins characterized by multiple copies of leucine rich repeats in the extracellular domain and IL-1 receptor motif in the cytoplasmic domain. Like its counterparts in Drosophila, TLRs signal through adaptor molecules (1). The TLR family is a phylogenetically conserved mediator of innate immunity that is essential for microbial recognition (2). Ten human homologs of TLRs (TLR1-10) have been described (3). Among this family of receptors, TLR2 and TLR4 have been most studied. These studies have suggested that TLR2 and TLR4 may serve as potential main mediators of LPS signaling (4,5). The TLR4 cDNA codes for a protein consisting of 799 amino acids with approximate molecular weight of 88 kDa (6).
Antigen
This antibody was developed by immunizing mice with Ba/F3 cell line expressing human TLR4 cell surface antigen (9).
Application Notes
The antibody has been shown to block the activation of monocytes with LPS (Paik, et al, 2003). The optimal condition should be determined for individual experiments.
Cell surface flow cytometric analysis of TLR4 in stable HEK293/hTLR4 cells using IMG-417A at 1 ug/10^6 cells. Secondary antibody Goat anti-Mouse IgG-PE (IMGENEX, 20103) at 0.25ug/10^6 cells. Both stable HEK293/hTLR4 and HEK293/Vector cell lines were equally stained.
Reference 1. Muzio M, Natoli G, Saccani S, Levrero M, and Mantovani A. J. Exp. Med. 187: 2097-2101 (1998). 2. Medzhitov R and Janeway CA. Cell 91: 295-298 (1997). 3. Chuang TH and Ulevitch RJ. Biochim. Biophys. Acta 1518 (1-2): 157-161 (2001). 4. Takeuchi O., Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa K, Takeda K and Akira S. Immunity 11: 443 (1999). 5. Poltorak A, Riccardi-Castagnoli P, Citterio S, and Butler B. Proc. Natl. Acad. Sci USA 97: 2163-2167 (2000). 6. Medzhitov R, Preston-Hurlburt P and Janeway CA Jr. Nature 388 (6640), 394-397 (1997). 7. Akashi S, Ogata H, Kirikae F, Kirikae T, Kawasaki K, Nishijima M, Shimazu R, Nagai Y, Fukudome K, Kimoto M, Miyake K. Biochem Biophys Res Commun. 268(1):172-177 (2000). 8. Shimazu, R., et al. MD-2, a Molecule that Confers Lipopolysaccharide Responsiveness on Toll-like Receptor 4. J. Exp. Med. 189(11): 1777-1782. 9. Paik YH, Schwabe RF, Bataller R, Russo MP, Jobin C, Brenner DA. Hepatology. 2003 May;37(5):1043-55.
Product Citations 1. Highly purified lipoteichoic acid activates neutrophil granulocytes and delays their spontaneous apoptosis via CD14 and TLR2. Lotz S, E Aga, I Wilde, G van Zandbergen, T Hartung, W Solbach, T Laskay. J. Leukoc. Biol. 75: 467-477 (2004). Imgenex antibodies cited [Flow (human PMN), Fig. 7B,C] : 1. TLR2 (IMG-416E) .2. TLR4 (IMG-417) . 2. Regulation of the Polymeric Ig Receptor by Signaling through TLRs 3 and 4: Linking Innate and Adaptive Immune Responses. Schneeman T, M Bruno, H Schjerven, F Johansen, L Chady, and C Kaetzel. J. Immunol. 175: 376-384 (2005). Imgenex antibodies cited [IF (HT29 human colon adenocarcinoma cell line), Fig. 2]: 1. TLR3 (IMG-315) 2. TLR4 (IMG-417) 3. Analysis of TLR4 Polymorphic Variants: New Insights into TLR4/MD-2/CD14 Stoichiometry, Structure, and Signaling. Rallabhandi P, J Bell, M Boukhvalova, A Medvedev, E Lorenz, M Arditi, V Hemming, J Blanco, D Segal, S Vogel.J. Immunol., 177: 322-332 (2006). IP, WB (HEK 293T cells), Fig. 3B. 4. Evidence of Toll-like receptor molecules on human platelets. Cognasse F, H Hamzeh, P Chavarin, S Acquart, C Genin, and O Garraud. Immunology and Cell Biology 83:196-198. (2205). Imgenex TLR antibodies cited [Intracellular Flow Cytomety and Cell Surface Flow Cytometry (human platelets), Figs. 1, 2]: 1. IMG-416D (TLR2-PE) 2. IMG-417D (TLR4-PE) 3. IMG-304A (TLR6) 4. IMG-321D (TLR8-PE) 5. IMG-305D (TLR9-PE) A comparison of staining results, intracellular versus cell surface flow cytometry is shown. 5. Intracellular Signaling Mechanisms Regulating Toll-Like Receptor-Mediated Activation of Eosinophils. Wong C, P Cheung, W Ip, C Lam. Am. J. Respir. Cell Mol. Biol 37: 85-96 (2007). Imgenex antibodies cited (human blood eosinophils and neutrophils from buffy coat): For WB, Fig. 1A: TLR1 (IMG-5012), TLR5 (IMG-664), TLR6 (IMG-304A), TLR7 (IMG-540), TLR8 (IMG-321A), TLR9 (IMG-305A). For Flow (Intracellular) and Flow (Surface), Fig. 1B: TLR1 (IMG-5021), TLR2 (IMG-416C), TLR3 (IMG-315C), TLR4 (IMG-417C), TLR5 (IMG-663C), TLR6 (IMG-304C), TLR7 (IMG-665A), TLR8 (IMG-321C), TLR9 (IMG-305C). 6. Cell Activation by Ligands of the Toll-Like Receptor and Interleukin-1 Receptor Family Depends on the Function of the Large-Conductance Potassium Channel MaxiK in Human Macrophages. Scheel O, M. Papavlassopoulos, R Blunck, A Gebert, T Hartung, U Zahringer, U Seydel, and A Schromm. Infection and Immunity 74: 4354-4356 (2006). IF/ICC (HEK293-TLR4/MD2-MaxiKGFP cells), Fig. 3. 7. Toll-Like Receptor Expression in Human Keratinocytes: Nuclear Factor B Controlled Gene Activation by Staphylococcus aureus is Toll-Like Receptor 2 But Not Toll-Like Receptor 4 or Platelet Activating Factor Receptor Dependent. Martin Mempel, Verena Voelcker, Gabriele Köllisch, Christian Plank, Roland Rad, Markus Gerhard, Christina Schnopp, Peter Fraunberger, Autar K. Walli, Johannes Ring, Dietrich Abeck, and Markus Ollert. J. Invest. Dermatol., 121: 1389-1396 (2003). Imgenex antibodies cited: 1. TLR2 (IMG-416) 2. TLR4 (IMG-417) [IF/ICC, Fig.2A and 2D (human keratinocytes)]. 8. Execution of Macrophage Apoptosis by PE_PGRS33 of Mycobacterium tuberculosis Is Mediated by Toll-like Receptor 2-dependent Release of Tumor Necrosis Factor-a. Basu S, SK Pathak, A Banerjee, S Pathak, A Bhattacharyya, Z Yang, S Talarico, M Kumdu, J Basu. J. Biol. Chem., 282 1039-1050 (2007). FA (human PBMC), Fig. 2C. 9. Expression of mRNA and proteins for toll-like receptors, associated molecules, defensins and LL-37 by SRIK-NKL, a CD8+ NK/T cell line. Srivastava M, B Srivastava. Leukemia Res. 29: 813-820 (2005). Imgenex antibodies cited [IF/ICC (human SRIK-NKL cells), Figs. 1, 2; Table 1.]: 1. TLR2 (IMG416) 2. TLR4 (IMG-417) 3. TLR7 (IMG-581) 4. TLR9 (IMG-305) 10. A cellular micro-RNA, let-7i, regulates toll-like receptor 4 expression and contributes to cholangiocyte immune responses against Cryptosporidium parvum infection. Chen X, P Splinter, S O'Hara, N LaRusso. J Biol Chem 282: 28929-28938 (2007). IF (human cholangiocyte cell lines), Fig. 5D.
11. RANTES modulates TLR4-induced cytokine secretion in human peripheral blood monocytes. Shahrara S, C Park, V Temkin, J Jarvis, M Volin, R Pope. J. Immunol. 177: 5077-5087 (2006). Imgenex antibodies cited: 1. TLR4 (IMG-417A): Flow (Cell Surface) [PB monocytes], Fig. 2C. 12. TLR2 and TLR4 expression on the immune cells of tuberculous pleural fluid. Prabha C, P Rajashree, D Sulochana. Immunology Letters 117: 26-34 (2008). Imgenex antibodies cited: 1. TLR2- FITC (IMG-416C): Flow (cell surface): Figs. 1A,B (human CD4+T cells, CD8+T cells, B cells, CD16+56+ cells and monocytes); 2(CD4+T cells); 4A,B (human Treg cells). Flow (intracellular): Fig. 3A,B (CD4+T cells) 2. TLR4- FITC (IMG-417C).Flow (cell surface): Figs. 1B,C (human CD4+T cells, CD8+T cells, B cells, CD16+56+ cells and monocytes); 2(CD4+T cells); 4A,B (human Treg cells). Flow (intracellular): Fig. 3A,B (CD4+T cells) 13.
Antibodies specific for human or murine Toll-like receptors detect canine leukocytes by flow cytometry. Burgener IA and TW Jungi. Veterinary Immunology and Immunopathology 124:184-191 (2008). Imgenex products cited for canine (dog) PBMC subpopulations:
14. TLR-independent induction of human monocyte IL-1 by phosphoglycolipids from thermophilic bacteria. Yang F, K Hua, Y Yang, W Zou, Y Chen, S Liang, H Hsu, S Wu. Glycoconjugate Journal: 10.1007/s10719-007-9088-2 (2007). WB (human TPH-1 monocytes), Fig. 5c. 15. The probiotic Escherichia coli strain Nissle 1917 induces γς T cell apoptosis via caspase-and FasL-dependent pathways.International Immunology 20:829-840 (2008). Imgenex antibodies cited: 1. IMG-416C (TLR2 FITC) Flow (cell surface), human PBMCs, Fig. 5. 2. IMG-417C (TLR4 FITC) Flow (cell surface), human PBMCs, Fig. 5. 16. Pro-osteogenic phenotype of human aortic value interstitial cells is associated with higher levels of toll-like receptors 2 and 4 and enhanced expression of bone morphogenetic protein 2. Yang X, D Fullerton, X Su, L Ao, J Cleveland, X Meng. Journal of the American College of Cardiology 53: 491-500 (2009). Imgenex antibodies cited: 1. IMG-416D (TLR2-PE): WB, Flow (cell surface): human aortic valve interstitial cells, Figs. 1A,B; 2C; 6A. 2. IMG-417D (TLR4-PE): WB, Flow (cell surface): human aortic valve interstitial cells, Figs. 1A,B; 2C; 6A. 17.
Stimulation of iNOS expression and apoptosis resistance in B-cell chronic lymphocytic leukemia (B-CLL) cells through engagement of Toll-like receptor 7 (TLR-7) and NF-kB activation. Hammadi A, Billard C, A-M Faussat, J-P Kolb. Nitric Oxide 19:138-145 (2008).Imgenex products cited (B-CLL cells from a leukemia patient):
1. IMG-417E (TLR4-FITC, clone HTA125): Flow (Intracellular), Fig 1: TLR4 was undetectable in the B-CLL cells.
2. IMG-305C (TLR9-FITC, clone 26C593.2): Flow (intracellular), Fig 1: TLR9 was detected in the BCLL cells
3. IMK-501 (Phospho-IkBa ActivELISA Kit): Fig 6B, ELISA data shows increased of IkBa phosphoryation following resiquimod treatment which stimulates NF-kB activation.
Note: No expression of TLR-4 or TLR-9 was detected at the surface of B-CLL cells by Flow (cell surface).
18. Functional heterogeneity and differential priming of circulating neutrophils in human experimental endotoxemia. Pillay J, B Ramaker, V Kamp, A Loi, S Lam, F Hietbrink, L Leenen, A Tool, P Pickkers, L Koenderman. J Leuko Bio 88: 221-220 (2010). TLR4 (IMG-417A): Flow (cell surface), human neutrophils and leukocytes, Fig 2. 19. High dose of bacterial LPS induce apoptosis-dependent tight junctional destruction in intestinal epithelial cells: Role of CD14. Wei-ting K, L Yu.http://ntur.lib.ntu.edu.tw/handle/246246/17800 (2008). The following monoclonal antibodies were used in flow (cell surface) cytometry: TLR4/CD284 (IMG-5031A) & TLR4/CD284 (IMG-417A). TLR4 was detected in T84 human colonadenocarcinoma cells but was not expressed in Caco-2 human colon carcinoma cells, various figures. 20. Human lymphatic endothelial cells express multiple functional TLRs. Pegu A, S Qin, B Junecko, R Nisato, M Pepper, T Reinhart. J Immunol 180: 3399-3405 (2008). The following antibodies were used in flow (cell surface and intracellular) cytometry: TLR2/CD282 PE (IMG-416D), TLR4 (IMG-417D), TLR5 FITC (IMG-663C), TLR6/CD286 FITC (IMG-304C), TLR9/CD289 PE (IMG-305D). Primary human lung microvascular endothelial cells, Fig 1C & 1D. Note: Data described but not shown for TLR9/CD289 PE (IMG-305D). 21. Toll like receptor expression and function in subsets of human T lymphocytes. Pietschmann K, S Beetz, S Welte, I Martens, J Gruen, H Oberg, D Wesch, D Kabelitz. Scandinavian J Immunol 70: 245-255 (2009). 1. TLR4/CD284 (IMG-417A): Flow (cell surface), Human T cell subsets (V sigma 1 and V sigma 2) isolated from PBMCs, Fig 3. 2. TLR6/CD286 (IMG-304A) & TLR8/CD288 PE (IMG-321D): Flow (intracellular), Human T cell subsets (V sigma 1 and V sigma 2) isolated from PBMCs, Fig 3. 22. In vitro response to Candida albicans in cultures of whole human blood from young and aged donors. Murciano C, E Vilamon, A Yanez, J Murciano, A Mir, D Gozalbo, M Gil. Immunol & Med Microbiol 51: 327-335 (2007). The following anitbodies were used in flow (cell surface) cytometry: TLR2/CD282 FITC (IMG-416C), TLR4 FITC (IMG-417C), TLR6/CD286 FITC (IMG-304C). Human whole blood from both young and elderly volunteers, Fig 1.
Note: The fluorescence mean intensity was measured in lymphocytes, monocytes, granulocytes (gated by their respective properties in forward and side scatter), Fig 1. 23. In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes. Giovanna Zanoni, Riccardo Navone, Claudio Lunardi, Giuseppe Tridente, Caterina Bason, Simona Sivori, Ruggero Beri, Marzia Dolcino, Enrico Valletta, Roberto Corrocher, Antonio Puccetti. PLoS Med 3(9): e358. DOI: 10.1371/journal.pmed.0030358 (2006). Imgenex antibodies cited- The anti-TLR4 monoclonal antibody clone HTA125, isotype IgG2b (Imgenex) was used..
Research purposes only.
Not for diagnostic or in vivo use. This product is guaranteed to perform as indicated on the datasheet for one year from the date of purchase.
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