Anti-TLR4 antibody [HTA125] - Low endotoxin, Azide free (ab30667)

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Immunogen is human TLR4-transfected mouse cell line Ba/F3, http://jem.rupress.org/content/189/11/1777.long

No epitope mapping has been performed, but based on application info for this antibody, it should be recognizing an extracellular epitope as no cell permeabilization is needed for flow cytometry. The extracellular domain is towards the N terminal and is more varied than the cytoplasmic domain at the C terminal; there are three isofrms of TLR4 whichdiffer in the first 200 amino acids and have varied molecular weights.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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The immunogen for ab30667, clone HTA125, was human TLR4-transfected mouse cell line Ba/F3, according to the paper describing development of the clone at the following URL, Shimazu et al, 1999:

http://jem.rupress.org/content/189/11/1777.long

Alignment of the amino acid sequences for human and bovine TLR4 (http://www.uniprot.org/uniprot/O00206 and http://www.uniprot.org/uniprot/Q9GL65 UniProt entries)

returns 76% identity. As I mentioned, if the epitope the antibody recognizes is conserved across both species, then we expect the antibody will cross-react, but there are many mismatches which the antibody would be unable to bind to on the bovine protein. Unfortunately, the epitope has not been mapped to a region with the the full-length sequence, which would allow a more precise assessment of potential for cross-reactivity.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for your inquiry.

I can confirm that the storage temperature of our products can be found on the individual datasheets.

Short term storage is usually two weeks. If the products is not used within two weeks of purchase, we in general recommend to aliquot it and to store as stated for the long term storage. As a general rule, freeze/thaw cycle are to be avoided.

Please find below the long term storage conditions for the products you are interested in:

ab9100 : Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.

ab3358 : Store at +4°C.

ab3359 : Store at +4°C.

ab30667 : Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.

ab86128 : Aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.

Since the vials are small and we want the text to be readable, we strongly suggest to check the datasheets for the storage information. This will have the positive side effect that also all the other valuable information on the datasheet will be seen.

I hope this information is helpful and wish you a sunny day.

Gracias por tu respuesta, y por compartir tus resultados con nosotros.

Es una lástima que este anticuerpo tampoco haya conseguido los resultados esperados.

Has probado ya dos lotes distintos de ab22048 y un lote de ab30677 sin éxito, se me ocurren algunas alternativas en un intento de ayudarte:

-Que nos mandes de nuevo el protocolo y lo revisemos por si hubiera alguna sugerencia que hacer (para ello adjunto el cuestionario, por si decidieras tomar esta opción). Si puedes envíanos también alguna imagen junto con el protocolo.

-Mandarte directamente de forma gratuita la última opción que queda contra esta proteína en Rata testado por WB (ab8376, que no esta actualmente en stock, tendríais que esperar entre una y dos semanas).

-Enviarte otro anticuerpo contra TLR4 no testado en WB o no testado en rata hasta el momento, pero con posibilidades de reaccionar (tendríamos que revisar las alternativas).

-Directamente reembolsarte el dinero del primer anticuerpo que adquiristeis (ab22048).

Siento mucho las molestias ocasionadas, y espero tu respuesta para que me digas por qué opción os inclináis.

Que pases una buena tarde.

Thank you for your question about ab30667. Generally, functional assays can cover a number of different experiments. However, this antibody has been used in blocking studies. As these will each be slightly different, we cannot provide a protocol, or give a recommended protocol. Instead we would recommend checking the literature for further information on using the same clone antibody (clone HTA125) in functional assays: 1) Shimazu, R. et al. (1999) MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll like receptor 4. J. Exp. Med. 189: 1777-1182.[http://www.ncbi.nlm.nih.gov/pubmed/10359581] 2) Yang, S. et al. (2001) Synergistic effect of muramyldipeptide with lipopolysaccharide or lipoteichoic acid to induce inflammatory cytokines in human monocytic cells in culture. Infect. Immun. 69: 2045-2053.[http://www.ncbi.nlm.nih.gov/pubmed/11254557] 3) Sugawara, S. et al. (2000). Proteolysis of human monocyte CD14 by cysteine proteinases (gingipains) from Porphyromonas gingivalis leading to lipopolysaccharide hyporesponsiveness. J. Immunol. 165: 411-418.[http://www.ncbi.nlm.nih.gov/pubmed/10861079] 4) Wang, J.E. et al. (2001) Involvement of CD14 and toll-like receptors in activation of human monocytes by Aspergillus fumigatus hyphae.[http://www.ncbi.nlm.nih.gov/pubmed/11254600] Infect Immun. 69: 2402-2406.[http://www.ncbi.nlm.nih.gov/pubmed/11254600] 5) Termeer, C. et al. (2002) Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4. J Exp Med. 195: 99-111.[http://www.ncbi.nlm.nih.gov/pubmed/11781369] I hope that this information is helpful. Please do not hesitate to contact us if you have further questions.