Toll-like receptors (TLR) are highly conserved throughout evolution and have been implicated in the innate defense to many pathogens. In Drosophila toll is required for the anti-fungal response, while the related 18-wheeler is involved in antibacterial defenses. In mammals, TLR identified as type I transmembrane signaling receptors with pattern recognition capabilities, have been implicated in the innate host defense to pathogens. TLR4 has been identified next to MD-2 and CD14 as a receptor that is central to the innate immune response to lipopolysaccharides (LPS) of Gram-negative bacteria. The HTA125 monoclonal antibody reacts preferentially, especially in flow cytometry, with human TLR4 (CD284) that is associated with MD-2. HTA125 is a TLR4 (CD284) function-blocking antibody that is useful for studies on the role of TLR4 (CD284) as a receptor for LPS induced cytokine production by TLR4 bearing cells. The antibody was shown to precipitate TLR4 (100 kDa). The antibody HTA125 is cross reactive with canine, cynomolgus monkey, rhesus monkey and marmoset monkey.
Immuno fluorescence, Immuno precipitation, Western blot
FC: 300000 cells/50 µl were stained with 2 µg antibody for 30 minutes at 4 °C
FS: In cell culture 10 µg/ml
IF: Oregon green labeled HTA125 was used in FRAP measurements
IP: HTA125 (4 mg/ml) coupled to Sepharose 4FF beads was added to cell lysate and incubated for 2 hours at 4 °C
W: 20 mg protein was analyzed on SDS-PAGE and transferred to nitrocellulose. Blot was blocked with TBS/5% dry milk/0.1% tween-20
For flow cytometry and immunohistology dilutions to be used depend on detection system applied. It is recommended that users test the reagent and determine their own optimal dilutions. The typical starting working dilution is 1:50.