Toll Receptor 12

The Toll-like receptor (TLR) multigene family encodes important recognition receptors of the innate immune system that have been conserved in both the invertebrate and vertebrate lineages . The mammalian host defense system is essentially regulated by these conserved Toll-like receptors.  At least 13 TLRs have been identified and cloned in mammalian cells which recognize molecular products/signals from all the major classes of pathogens and activation of innate immunity.  TLRs were identified as genes coding for both an N-terminal leucine-rich repeat (LRR) domain and a C-terminal Toll-IL-resistance (TIR) domain. The Toll signaling to NF-Kb starts form conserved Toll-IL-1-resistance (TIR) domain, which mediated the coupling of TIR adaptor molecules (MyD88, Mal, TICAM and TRAM) and caused production of inflammatory cytokines such as IL-1, IL-6, IL-8 , TNFa, and IL-12, chemokines and co-stimulatory molecules such as CD40, CD80 and CD86.  In the presence of inflammatory cytokines and binding of adaptor molecule, MyD88 that binds FADD and triggers apoptosis through the caspase cascade.  TLR induced apoptosis pathway appears to be a repertoire of defense mechanism utilized by innate defense mechanism.  The constitutive expression of many human TLRs (1, 2, 3) have been shown on the surface of myeloid lineage cells by RT-PCR and use of specific monoclonal antibodies.  Upon activation of these receptors by their respective chemokines and ligands have been shown in literature on various cell lines including endothelial, epithelial and other cells.  The expression of TLR 3, 7, 8 and 9 are mainly found on endosomal lysosomal compartments.  Human TLR3 is expressed in human fibroblasts cells and TLR 9 in in-vitro derived DC cells.   There is significant evidence of TLR involvement is many systemic disorders following bacterial infection including sepsis, peridontitis, cardiac ischemia, cerebral palsy and others, understanding the TLRs involvement in these conditions will allow therapeutic interventions at the receptor level for treatment of these disorders. 

The TLR are highly conserved protein and share structural and functional domains across species.  These receptors recognize pathogen associated molecular patters (PAMPs) that are expressed on infectious agents, and mediate the production of cytokines needed for production of immediate immunity. The cell-surface TLRs, including TLR1, TLR2, TLR4, and TLR6, recognize microbial membrane lipids, whereas TLR3, TLR7, TLR8, and TLR9 recognize pathogen-derived nucleotides in intracellular compartments.  TLR7 and TLR9 respond to host-derived nucleotides as well, and they have been implicated in a variety of autoimmune diseases. Toll-like receptor 9 (TLR9) is a receptor for unmethylated CpG dinucleotides found in bacterial and viral DNA.  TLR11-13 and TLR21-23 subfamilies are represented by pseudogenes in humans. 

TLR11-13 are important targets in central nervous system infection. The TLR12 receptors recognize uropathogenic bacteria antigens (4).    TLR12 is a membrane bound protein with two TMD.  The first human toll like receptor to be identified, TLR4, senses lipopolysaccharide (LPS) while TLR2 on the other hand senses diacylated or triacylated lipopeptides after heterodimerizing with either TLR6 or TLR1, respectively.  The conserved cytoplasmic TIR (Toll/IL-1 receptor) domains of the IL-1 and Toll-like receptors are the critical focal point for the generation of ligand-induced cytoplasmic signaling cascades. For signaling all the TLRs utilize one or more of the four known TIR-containing adaptor molecules: MyD88, TIRAP/MAL, TRIF, and TRAM.  Human TLR9 receptor is expressed as A and B isoforms with apparent MW 125kDa (1032 aa) and 120kDa (975) with several splice variants.  TLR antibody will label both A and B isoforms. TLR9 is a trans-membrane receptor protein characterized by extracellular domains with leucine-rich repeats and a cytoplasmic domain with type I IL-1 receptor.  The TLR11is expressed on cell surface of peripheral blood mononuclear cells along with major histocaompatibility comples (MHC) class II CD19 positive cells.  Many specific adapter molecules (TICAM, MyD88, ICAM2, TRAM, TIRAP, TRIF) are involved in signaling of TLRs.

The TLR 12-selective antibodies were generated against peptide form unique region form the mid-region (aa 444-465) of Toll receptor-12 protein, this peptide sequence is not present in other members of the TLR family.    FabGennix Inc. has generated rabbit anti-TLR12 mono-epitope-specific antibodies utilizing linear and cyclic peptide methodology.  The Anti-TLR12 antibodies have been fully characterized for cross reactivity with other members of the TLR family molecules and with cellular proteins using Western blot analyses.  TLR12 antibody is purified on immobilized antigen affinity based chromatography and stabilized for long-term storage.  The TLR12 antibody is also available as FITC-conjugate, other conjugates can be ordered at a nominal charge.  FabGennix International Inc., has produced antibodies to all Toll receptors (TLR1-TLR13), these antibodies are now available for sale from us or from our distributors.   FabGennix Int. Inc., also provide western blot positive controls for TLR11 in ready-to-use buffer.  Limited quantities of the antigenic blocking peptide for TLR12 antibodies is also available (inquire for availability).

Immunogen:  Synthetic peptides are selected from human Toll receptor 12 (TLR12) corresponding to amino acids 444-465 form the amino terminal end of mouse  TLR12 protein, the peptide was post-synthetically modified to achieve desired antigenicity before coupling to a carrier protein.  

Concentration: TLR-1201AP antibody has Ig concentration ranging from 0.55-0.69mg/ml in antibody stabilization buffer. 

Applications: For ELISA/Dot blot assay the antibody TLR-1101AP can be diluted to 1:20,000 in diluObuffer (Cat # FGI-1963).  For Western blot applications starting dilution of 1:500 in DiluObuffer (Cat # FGI-1963) is recommended.  Other applications and species cross reactivity for this antibody is yet to be determined.      

Reactivity:      The antibody TLR-1201AP labels a diffused band of around 94kDa in PC-TLR12 samples.  The Western on the left is a 11% gel with antibody TLR-1201AP diluted 1:500 in diluObuffer. 

Protocols:       Standard protocol for various applications (Western blot; immunoprecipitation and immunohistochemistry) of this antibody can be obtained by calling technical service hotline. The general information on this antibody is supplied with the product.  FabGennix Inc. recommends investigators to optimize conditions for optimal usage of this product.   

Storage:            The antiserum is supplied in antibody stabilization buffer with preservatives.  For long-term storage of antibody, store at -20^oC FabGennix Inc. does not recommend storage of very dilute antibody solutions unless they are prepared in specially formulated multi-use antibody dilution buffer (Cat # DiluOBuffer).  Working solutions of antibodies in DiluOBuffer should be filtered through 0.45um filter after every use for long-term storage.

Notes:           Briefly centrifuge to collect liquid, heat or boil PC-TLR12 tube for 1-2 minutes to dissolve any precipitate before use.  This product is “ready-to-use” for electrophoresis. After thawing store at room temperature, Repeated freezing and thawing may result in appearance of higher MW immunoreactive bands.   

 

For research use only, not for diagnostic or therapeutic use.