Overview

  • Product name
    Anti-TLR4 antibody [HTA125]
    See all TLR4 primary antibodies
  • Description
    Mouse monoclonal [HTA125] to TLR4
  • Host species
    Mouse
  • Specificity
    This antibody recognises the human Toll like receptor 4 (TLR4) cell surface antigen. TLR4 has been demonstrated to act as alpha receptor for LPS on human monocytes and macrophages. TLR4 signalling of LPS stimulation requires the presence of the MD-2 molecule.TLR4 is weakly expressed by resting cells, but is upregulated following stimulation with LPS. We do not guarantee WB since we receive variable results from our customers and cannot make this antibody work in our hands in WB in denaturing and reducing conditions. The immunogen is a full-length immunogen so we suspect that to get the product to work in WB, non-reducing or native WB could be a better option. THP1 monocytes or macrophage are good positive controls and HeLa/Hek293 should be suitable negative/low expression controls. Membrane preparation may be better that whole cell as expression levels may not be that high in some samples. We welcome any further feedback in WB by participating to our AbTrial program.
  • Tested applications
    Suitable for: Flow Cyt, IPmore details
  • Species reactivity
    Reacts with: Rat, Guinea pig, Human, Pig, Rhesus monkey
  • Immunogen

    Ba/F3 cell line expressing TLR4

Properties

Applications

Our Abpromise guarantee covers the use of ab8376 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 - 1/25.

Use 10ul of the suggested working dilution to label 106 cells or 100ul whole blood. Method sheets are available on request.

 

 

 

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

 

IP Use at an assay dependent concentration.

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

  • Staining of human peripheral blood monocytes with ab8376.

References

This product has been referenced in:
  • Cao Z  et al. Tissue expression of Toll-like receptors 2, 3, 4 and 7 in swine in response to the Shimen strain of classical swine fever virus. Mol Med Rep 17:7122-7130 (2018). Read more (PubMed: 29568891) »
  • Peng J  et al. High-Throughput Sequencing and Co-Expression Network Analysis of lncRNAs and mRNAs in Early Brain Injury Following Experimental Subarachnoid Haemorrhage. Sci Rep 7:46577 (2017). Read more (PubMed: 28417961) »
See all 16 Publications for this product

Customer reviews and Q&As

1-10 of 11 Abreviews or Q&A

Answer

Thank you for contacting us. I am sorry to hear this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.

Having reviewed the protocol details, I believe this product should have given satisfactory results. I apologize for the inconvenience and am pleased to offer you a credit note/ refund in compensation.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

PS: Please note as the refund or credit note will be given so theimage discount code provided for this antibody will not be valid.

Read More

Answer

Thank you for your email.

I have checked the images and based on the images and protocol, I have following suggestions;

- Use a different HRP conjugated antibody. DAKO ChemMate EnVision Detection Kit as per DAKO website is optimized for IHC only. You might need to check whether the antibody DAKO ab is suitable for western blot. Please check with DAKO also.

- Try heating the lysates 70C for 15-20 minutes. As TLR4 is a single pass membrane protein so heating at low incubation temperature for longer period of time would help.

- The background staining can be reduced using 5% BSA as an alternative to milk. Please try BSA.

- Please check and optimize the tissue homogenization/ protein preparation protocol.

- Please use RAW or Daudi cell lysates as positive control or cell lysates other than the TCA prepared lysates.

I hope these suggestions would help. Should the results do not improve please let me know I will be happy to replace the antibody.

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Answer

Thank you very much sending details.

I am surprised to hear that antibody failed in 2 different occasions as we haven't received any complaint for these lots so I think protocol might need further optimizations. Could you provide image of staining? Could you provide how you have prepared the samples and how old the samples were?

I will look forward to hearing from you soon.

Read More

Question

Recap of the complaint

Date(s) submitted: 13-Jun-12

Catalog number(s): 8376

Product(s): Mouse monoclonal [HTA125] to TLR4

CCEID: 3850619
Survey results
Telephone section
Q. Did you contact Abcam's Scientific Support by telephone in reference to this complaint?
A. Yes
Q. Was it clear from the phone options which number you needed to press to get your issue resolved?
A. Yes

Protocol advice section
Q. Did Abcam's Scientific Support give you protocol advice?
A.No
Q. How did you feel about being given protocol advice?
A.
Q. Did the product work successfully after following the advice?
A.
Q. Why have you not tried the protocol advice? (Select all that apply.)A.

Free of charge replacement section
Q. Did Abcam's Scientific Support give you a free of charge replacement?
A. Yes

Q. Did the replacement product work successfully?
A.No
Credit note / refund section
Q. Did Abcam's Scientific Support give you a credit note or a refund?
A.No

Q. Would you have preferred a different outcome from receiving a credit note or refund?
A.
Overall satisfaction section
Q. How satisfied are you with how the complaint was handled?
A.Unsatisfied
Q. Could we have done anything to better resolve the problem?
A.Could you provide any other suggestion to make TLR4 WB work or give a credit note (to order different antibody) ?
Q. Would you mind if our scientific support team contacted you to better understand what happened and reach an agreeable solution?
A.No, I would not mind
Q. Please provide a phone number or email address where we can reach you.
Q. Please share with us any final thoughts you may have about Abcam, the complaints process, or our products.
A.appreciate this sort of following up

Read More
Answer

Thank you for completing the survey on Abcam website.

I am sorry to hear that the antibody did not work with porcine sample. I have attached a questionnaire, to follow up for the complaint. Could you complete this and send it to me asap. I will then check if troubleshooting of protocol could help.

Many thanks for your cooperation in advance. I will look forward to hearing from you soon.

Read More

Question
Answer

Thank you for your email.

The order number I have created is 1107878; you will be receiving this antibody shortly.

TLR4 is highly expressed in porcine kidney so the antibody should have worked. Please let me know if you experience any problem again.

Could I recommend using human, kidney, liver, placenta or spleen tissue lysates as positive control lysates, just to double check if the porcine lysates are OK.

Read More

Answer

Thank you for contacting us.

I am sorry, I forgot to ask the order number of your last purchase. Could you send the purchase order number to us so that we can send the new vial at same address?

Thanks! Looking forward to hearing from you soon.

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Question
Answer

DISCOUNT CODE: XXXXXXXXXXX (for ab8376)
DISCOUNT CODE: XXXXXXXXXXX (for ab11354)

Expiration date: 24-09-2012

I am very pleased to hear you would like to use ab8376 (WB) and ab11354(IHC-P) inPorcine and send us an image of your results.

This code will give you2 free [PRIMARY ANTIBODY/PROTEIN] before the expiration date. To redeem this offer, please submit an Abreview with the image for [IHC-P and WB] and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active.
For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

The code will be active once the Abreview with the image has been submitted and can be redeemed in one of the following ways:
1) Call to place your order and mention the code to our customer service department;
2) Include the code in your fax order;
3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated. Remember if the product does not work you will be covered by our Abpromise guarantee and eligible for a full refund or replacement.

If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

Read More

Answer

Thank you for your reply. I'm afraid all the information we have is from those references and therefore we do not have any more data looking at the blocking ability of the antibody. We welcome all feedback on our antibodies via our Abreviews system and I hope you will consider sharing your data with other users of the antibody; all feedback is rewarded with Abpoints which can be used for gifts and discounts. I'm sorry I couldn't help you more on this occasion and wish you all the best with your research,

Read More

Answer

I can confirm that the blocking activity of this clone has been established in a number of studies, for your convenience I have listed some useful articles below: 1: Infect Immun. 2001 Apr;69(4):2045-53. Synergistic effect of muramyldipeptide with lipopolysaccharide or lipoteichoic acid to induce inflammatory cytokines in human monocytic cells in culture. Yang S, Tamai R, Akashi S, Takeuchi O, Akira S, Sugawara S, Takada H. 2: J Exp Med. 2002 Jan 7;195(1):99-111. Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4. Termeer C, Benedix F, Sleeman J, Fieber C, Voith U, Ahrens T, Miyake K, Freudenberg M, Galanos C, Simon JC. 3: Infect Immun. 2001 Apr;69(4):2402-6. Involvement of CD14 and toll-like receptors in activation of human monocytes by Aspergillus fumigatus hyphae. Wang JE, Warris A, Ellingsen EA, Jorgensen PF, Flo TH, Espevik T, Solberg R, Verweij PE, Aasen AO. 4: J Immunol. 2000 Jul 1;165(1):411-8. Proteolysis of human monocyte CD14 by cysteine proteinases (gingipains) from Porphyromonas gingivalis leading to lipopolysaccharide hyporesponsiveness. Sugawara S, Nemoto E, Tada H, Miyake K, Imamura T, Takada H. 5: J Exp Med. 1999 Jun 7;189(11):1777-82. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-likereceptor 4. Shimazu R, Akashi S, Ogata H, Nagai Y, Fukudome K, Miyake K, Kimoto M. Please note that the references and datasheet mention the clone HTA125, which should be used Azide free (or using ab30667 which is supplied with no Azide). I wonder therefore if the lack of blocking you observe could therefore be due to the presence of Azide. I hope the above references will help you,

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Question
Answer

Unfortunately I do not believe that the epitope that this antibody recognises has been fully characterised and I have been unable to locate any further information relating to the terminus recognised. I am sorry that I cannot be of additional assistance in this matter,

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1-10 of 11 Abreviews or Q&A

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