Overview

  • Product name
    Anti-TLR4 antibody [HTA125] - Low Endotoxin
    See all TLR4 primary antibodies
  • Description
    Mouse monoclonal [HTA125] to TLR4 - Low Endotoxin
  • Host species
    Mouse
  • Specificity
    ab30667 recognises the human Toll like receptor 4 (TLR4) cell surface antigen. This antibody has been demonstrated to block activation of monocytes with LPS. TLR4 expression levels and cleavage or degradation products can vary between different cell and tissue samples. Customers have observed this variability in WB band size and our laboratory has confirmed this variability as well observing lower molecular weight cleavage and degradation products and in some samples a lack of the full length TLR4 band. The TLR4 cleavage and degradation products and potential lack of full length TLR4 are well documented in the literature, including PMID 16885150 and 22927440. We recommend running a positive control human intestine tissue lysate.
  • Tested applications
    Suitable for: Flow Cyt, IP, Blocking, ICC/IFmore details
  • Species reactivity
    Reacts with: Rat, Human, Rhesus monkey
  • Immunogen

    Ba/F3 cell line expressing TLR4.

  • General notes
    Endotoxin Level: <0.01 EU/ug

Properties

Applications

Our Abpromise guarantee covers the use of ab30667 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Flow Cyt 1/10 - 1/50.

Use 10µl of the suggested working dilution to label 106 cells or 100µl whole blood.
ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

IP Use at an assay dependent concentration.
Blocking Use at an assay dependent concentration. PubMed: 17767165
ICC/IF Use at an assay dependent concentration. PubMed: 17916800

Target

  • Function
    Cooperates with LY96 and CD14 to mediate the innate immune response to bacterial lipopolysaccharide (LPS). Acts via MYD88, TIRAP and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response. Also involved in LPS-independent inflammatory responses triggered by Ni(2+). These responses require non-conserved histidines and are, therefore, species-specific.
  • Tissue specificity
    Highly expressed in placenta, spleen and peripheral blood leukocytes. Detected in monocytes, macrophages, dendritic cells and several types of T-cells.
  • Involvement in disease
    Genetic variation in TLR4 is associated with age-related macular degeneration type 10 (ARMD10) [MIM:611488]. ARMD is a multifactorial eye disease and the most common cause of irreversible vision loss in the developed world. In most patients, the disease is manifest as ophthalmoscopically visible yellowish accumulations of protein and lipid that lie beneath the retinal pigment epithelium and within an elastin-containing structure known as Bruch membrane.
  • Sequence similarities
    Belongs to the Toll-like receptor family.
    Contains 18 LRR (leucine-rich) repeats.
    Contains 1 LRRCT domain.
    Contains 1 TIR domain.
  • Domain
    The TIR domain mediates interaction with NOX4.
  • Post-translational
    modifications
    N-glycosylated. Glycosylation of Asn-526 and Asn-575 seems to be necessary for the expression of TLR4 on the cell surface and the LPS-response. Likewise, mutants lacking two or more of the other N-glycosylation sites were deficient in interaction with LPS.
  • Cellular localization
    Membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • ARMD10 antibody
    • CD284 antibody
    • CD284 antigen antibody
    • Homolog of Drosophila toll antibody
    • hToll antibody
    • TLR 4 antibody
    • TLR4 antibody
    • TLR4_HUMAN antibody
    • TOLL antibody
    • Toll like receptor 4 antibody
    • Toll-like receptor 4 antibody
    see all

Images

  • ab30667 staining TLR4 in human peripheral blood monocytes by Flow Cytometry analysis.
  • ICC/IF image of ab30667 stained rat adrenal medulla PC12 cells. The cells were 4% PFA and 100% Methanol fixed (10 min) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab30667, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue).

References

This product has been referenced in:
  • Liu W  et al. MicroRNA-146a suppresses rheumatoid arthritis fibroblast-like synoviocytes proliferation and inflammatory responses by inhibiting the TLR4/NF-kB signaling. Oncotarget 9:23944-23959 (2018). Read more (PubMed: 29844864) »
  • Yokoi T  et al. Inhibition of Toll-like receptor 4 ameliorates experimental postischemic injury in the cholestatic liver through inhibition of high-mobility group box protein b1 (HMGB1) signaling. Surgery 163:270-276 (2018). IHC-P . Read more (PubMed: 29191677) »
See all 35 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Answer

Thank you for contacting us.
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.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Question
Answer

Thank you for contacting us.

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.

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Answer

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.

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Answer

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.

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Answer

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.

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Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Specification
HeLa
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.5% Triton X100 in PBS

Dr. Kirk Mcmanus

Verified customer

Submitted Feb 25 2009

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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