Description: The HTA125 monoclonal antibody reacts with human Toll-like receptor 4 (TLR4). So far, at least ten members of the Toll family have been identified in humans. This family of type I transmembrane proteins is characterized by an extracellular domain with leucine-rich repeats and a cytoplasmic domain with homology to the type I IL-1 receptor. Two of these receptors, TLR2 and TLR4, are pattern recognition receptors and signaling molecules in response to bacterial lipoproteins and have been implicated in innate immunity and inflammation. TLR4 physically associates with another molecule called MD-2, and together with CD14, this complex is responsible for LPS recognition and signaling. TLR4 is expressed by peripheral blood monocytes. HTA125 has been reported to immunoprecipitate human TLR4 (~100 kDa) from transfected cells. Most TLR cell surface expression, especially TLR1 and TLR4, occurs at low levels on monocytes and at even lower levels on other cell types including granulocytes and immature dendritic cells (iDC). Furthermore, a relatively high degree of variability in TLR surface expression has been reported among normal donors.
It is highly recommended that for optimal staining of TLR4, whole blood be stained using the lysed whole blood protocol (found in Best Protocols) rather than Ficoll-gradient prepared normal human peripheral blood cells. The use of a density gradient appears to reduce the staining intensity significantly.
Applications Reported: The HTA125 antibody has been reported for use in flow cytometric analysis, and immunoprecipitation. It has also been reported in blocking of LPS-induced cytokine production. For detection of peripheral monocytes, a three step staining protocol is recommended using purified anti-human TLR4 followed by biotin anti-mouse IgG and streptavidin-PE. (Please use Functional Grade purified HTA125, cat. 16-9917, in functional assays.).
Applications Tested: The HTA125 antibody has been tested by flow cytometric analysis of normal human peripheral blood cells. This can be used at less than or equal to 1 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test.
Purity: Greater than 90%, as determined by SDS-PAGE.
Aggregation: Less than 10%, as determined by HPLC.
Filtration: 0.2 µm post-manufacturing filtered.
TLR4 is 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. This receptor is most abundantly expressed in placenta, and in myelomonocytic subpopulation of the leukocytes. Mammalian cells respond to LPS by activating TLR4. TLR4 belongs to the multi-protein complex of lipopolysaccharide (LPS) receptor, containing CD14, LY96 and TLR4, and is involved in signal transduction events induced by lipopolysaccharide (LPS) found in most gram-negative bacteria. TLR4 aids in the recognition of 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. Mutations in the TLR4 gene have been associated with differences in LPS responsiveness. Also, several transcript variants of the TLR4 gene have been found, but the protein coding potential of most of them is uncertain. TLR4 is expressed by peripheral blood monocytes and a small population of B-cells and is also expressed in human placenta. Studies with TLR4-deficient mice indicate that the main ligand for TLR is lipopolysaccharide. Consequently, these mice also showed increased susceptibility to Gram-negative sepsis.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Protein Aliases: CD284; homolog of Drosophila toll; hToll; Toll-like receptor 4
Gene Aliases: ARMD10; CD284; TLR-4; TLR4; TOLL
UniProt ID: (Human) O00206
Entrez Gene ID: (Human) 7099