Overview

  • Product name
    Anti-TLR3 antibody [40C1285]
    See all TLR3 primary antibodies
  • Description
    Mouse monoclonal [40C1285] to TLR3
  • Host species
    Mouse
  • Specificity
    Negative results in WB have been obtained for mouse interstine lysates (human intestine is a positive control). The protein is heavily glycosylated and the observed band size should be higher than the predicted 103kDa. The protein is found at different molecular weight in different tissues so optimization may be necessary.
  • Tested applications
    Suitable for: IHC-P, WB, Flow Cyt, IPmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide:

    VLNLTHNQLRRLPAAN

    conjugated to KLH, corresponding to amino acids 55-70 of Human TLR3

  • Positive control
    • Human intestine tissue lysate, mouse spleen

Properties

Applications

Our Abpromise guarantee covers the use of ab13915 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
IHC-P Use a concentration of 5 - 10 µg/ml. Permeabilization of samples might be necessary and overnight incubation with the ab13915 is recommended.
WB Use a concentration of 1 - 3 µg/ml. Detects a band of approximately 130 kDa (predicted molecular weight: 108 kDa). TLR3 is a single-pass type I membrane protein. We recomend not to boil the samples, instead heat lysates at 60-70C for 10 minutes.
Flow Cyt Use 2-5µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

IP Use 2µg for 106 cells.

Target

  • Function
    Key component of innate and adaptive immunity. TLRs (Toll-like receptors) control host immune response against pathogens through recognition of molecular patterns specific of microorganisms. TLR3 is a nucleotide-sensing TLR which is activated by double-stranded RNA, a sign of viral infection. Acts via MYD88 and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response.
  • Tissue specificity
    Expressed at high level in placenta and pancreas. Also detected in CD11c+ immature dendritic cells. Only expressed in dendritic cells and not in other leukocytes, including monocyte precursors. TLR3 is the TLR that is expressed most strongly in the brain, especially in astrocytes, glia, and neurons.
  • Involvement in disease
    Defects in TLR3 are associated with herpes simplex encephalitis type 2 (HSE2) [MIM:613002]. HSE is a rare complication of human herpesvirus 1 (HHV-1) infection, occurring in only a small minority of HHV-1 infected individuals. HSE is characterized by hemorrhagic necrosis of parts of the temporal and frontal lobes. Onset is over several days and involves fever, headache, seizures, stupor, and often coma, frequently with a fatal outcome. Note=TLR3 mutations predispose otherwise healthy individuals to isolated herpes simplex encephalitis through a mechanism that involves impaired IFNs production and reduced immune defense against viral infection in the central nervous system.
  • Sequence similarities
    Belongs to the Toll-like receptor family.
    Contains 22 LRR (leucine-rich) repeats.
    Contains 1 LRRCT domain.
    Contains 1 LRRNT domain.
    Contains 1 TIR domain.
  • Domain
    ds-RNA binding is mediated by LRR 1 to 3, and LRR 17 to 18.
  • Post-translational
    modifications
    Heavily N-glycosylated, except on that part of the surface of the ectodomain that is involved in ligand binding.
  • Cellular localization
    Endoplasmic reticulum membrane. Endosome membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • CD283 antibody
    • CD283 antigen antibody
    • IIAE2 antibody
    • TLR 3 antibody
    • Tlr3 antibody
    • TLR3_HUMAN antibody
    • Toll Like Receptor 3 antibody
    • Toll-like receptor 3 antibody
    see all

Images

  • All lanes : Anti-TLR3 antibody [40C1285] (ab13915) at 3 µg/ml

    Lane 1 : Untransfected HEK293 cell lysate
    Lane 2 : HEK293 cell lysate transfected with human TLR3 cDNA whole cell lysate
    Lane 3 : Human intestine tissue lysate
    Lane 4 : Human placenta tissue lysate
    Lane 5 : Human heart tissue lysate
    Lane 6 : Human ovary tissue lysate

    Secondary
    All lanes : Goat anti-mouse HRP conjugate

    Predicted band size: 108 kDa

  • Immunohistochemical analysis of formalin fixed paraffin embedded tissue section of mouse intestine using ab13915 at 1/500 dilution with HRP-DAB detection and hematoxylin counterstaining. The image shows a punctate staining of the ER and endosomes in a subset of cells in Peyer's patches (organized lymphoid nodules) in the tested section.

  • Flow cytometry analysis of human B cells using ab13915 at 1µg/106 cells followed by FITC conjugated secondary antibody (Red). Green shows isotype control.  

  • Western blot analysis of TLR3 in lysates from untransfected 293 cells (lane A), 293 cells transfected with human TLR3 cDNA (lane B), and 20 µg/lane human intestine tissue lysate (lane C).

    Western blot analysis of TLR3 in lysates from untransfected 293 cells (lane A), 293 cells transfected with human TLR3 cDNA (lane B), and 20 µg/lane human intestine tissue lysate (lane C).
  • Immunohistochemical analysis of formalin fixed paraffin embedded tissue section of mouse colon using ab13915 at 1/500 dilution with HRP-DAB detection and hematoxylin counterstaining. Intense signal was found in subset of cells at the bases of the crypts.

  • ab13915 at 2µg / 1x106 cells staining intracellular TLR3 in human monocytes by Flow Cytometery. A PE-conjugated goat polyclonal to mouse IgG1 was used as secondary. The shaded histogram repersents monocytes without antibody; green repersents isotype control and red histogram shows staining with ab13915. 

  • ab13915 at 5ug/ml staining TLR3 in mouse spleen tissue section by Immunohistochemistry (Formalin/ PFA fixed paraffin-embedded sections). The left image repersents staining with isotype control and right one repersents staining with ab13915.

  • ab13915 staining TLR3 in SW480, SW620 (colon cancer) and FaDu, Detroit 562 obtained from human cells by Flow Cytometry. Samples were prepared by trypsinizining and washing in PBS and centrifugation in a buffer containing 2% FBS, 0.1% Na-azide and PBS. Cells were fixed in paraformaldehyde and permeabilized in 0.2% Tween. Gating and analysis was performed by WinMDI 2.9 software. The primary antibody was diluted to 1 µg/cells (PBS, 2% FBS and 0.1% Na-azide) and incubated with sample for 30 minutes at 4°C. An FITC conjugated rabbit polyclonal to mouse IgG, diluted to 1/40, was used as secondary. Image represents FACS analysis of TLR3 protein expression in SW480 (A), SW620 (B), FaDu (C) and Detroit 562 (D) cell lines. Cell lines were permeabilized and immunostained with TLR3-specific antibody (thick black l

    See Abreview

References

This product has been referenced in:
  • Ni YH  et al. Distinct expression patterns of Toll-like receptor 7 in tumour cells and fibroblast-like cells in oral squamous cell carcinoma. Histopathology 67:730-9 (2015). IHC . Read more (PubMed: 25828894) »
  • Allman WR  et al. TACI deficiency leads to alternatively activated macrophage phenotype and susceptibility to Leishmania infection. Proc Natl Acad Sci U S A 112:E4094-103 (2015). Mouse . Read more (PubMed: 26170307) »
See all 13 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Application
Western blot
Loading amount
25 µg
Gel Running Conditions
Reduced Denaturing
Sample
Mouse Tissue lysate - whole (liver)
Specification
liver
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Dec 01 2014

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 20 minute(s) · Concentration: 1% · Temperature: 25°C
Sample
Mouse Cell (Macrophages)
Specification
Macrophages
Permeabilization
Yes - Triton X 0.1% 5 min
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Jul 23 2014

Answer

Thank you for contacting us.

Unfortunately the epitope for these two antibodies has not been mapped, and therefore I am unable to provide the sequence they recognise.

However, ab13915 is currently in stock. If this antibody gave you good results I would recommend you to continue to use it. Ab13915 is a very good antibody.

We do have some other anti TLR3 antibodies validated for IHC-P. You can have a look at the list by https://www.abcam.com/Search?Keywords=TLR3+&fReset=1&pt=1&source=TopSearch&btnSearch=Search#Search?AppliedFacets.FacetProductType=Primary+antibodies&AppliedFacets.FacetTargetName=TLR3&AppliedFacets.FacetApplication=IHC-P&IsFacetsOnly=False&PageNumber=1&Keyw.

I hope this information is helpful to you. If not please let me know and I will be happy to assist you further.

Read More

Answer

Thank you for contacting us. This antibody has not been tested in frozen sections or in cells, or in paraffin embedded tissue sections. Typically though, antibodies that work well in paraffin embedded sections also work in frozen tissues.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Question

Kindly advise in regards to the following information. The images are attached.
Antibody code: ab13915, ab22048, ab134368
details:
Ab13915: Po: 89238 Lot: GR52440 Invoice: 1345585
Ab22048: Po: 89238 Lot: GR77209 Invoice: 1345585
Ab134368: Po: 89238 Lot: GR95073 Invoice: 1346631
Antibody storage conditions (temperature/reconstitution etc): for ab13915 and ab22048 at -20- -80ºC ,
for ab134368 at 4 ºC
Description of the problem (high background, wrong band size, more bands, no band etc.): too many nonspecific band that could not allow me to determine my band
Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)
Purified proteins from human cell lines
Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)
RIPA buffer with protease inhibitors and heating the samples for 10mins at 70 ºC
Amount of protein loaded
Out of the transfected cell 5yg and 20ug were beingloaded, out of the cell lines 100ug were loaded
Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
Reducing gels. 4-12% and 3-8%
Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)
Transfer buffer: glycin, trisma base and methanol, transfer time: 45min for one membrane. Blocking: 5% skim milk, also for ab13915 I tried3% BSA. I tried 1.5hrs blocking and I tried overnight only for ab13915
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step): Abcam, they all react with human, mouse. The antibody was diluted in PBS with 0.02% NA-azide (also I tried 3% BSA for ab13915). Dilution used was: 2ug/ml for ab13915, Dilution used was: 3ug/ml for ab22048, Dilution used was: 3ug/ml for ab134368. Incubation time was overnight at 4 ºC and also for ab13915 I tried 1.5hrs.
Three washings for 5mins with PBST
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step):
Dako, HRP-antimouse, diluted 1:60 in %5 blocking skim milk for 20mins, followed by three washes with PBST 5mins each with shaking
Detection method (ECL, ECLPlus etc.): ECL
Positive and negative controls used (please specify): for ab13915 transfected and untrasnfected cells with TLR3 plamis (pEF6/V6-His-TOPO, for ab22048 THP1 cell lines which are known to express TLR4, ab134368 transfected and untrasfected cells withTLR9 plasmid PcDNA3
Optimization attempts (problem solving)
How many times have you tried the Western? Three to four times
Have you run a "No Primary" control? No
Do you obtain the same results every time? e.g. are the background bands always in the same place? Yes
What steps have you altered? I tried to t do overnight blocking, to do Blocking with BSA, to incubate the primary antibody for 1.5hrs only rater than overbight,..
Thanks in advance for your assistance.
Kind Regards,

Read More
Answer

Thank you for your email. I am sorry to hear that you have been experiencing problems with this antibody.
I have read the details you have kindly provided and have following further suggestions;
- Try loading 30-40 ug of protein lysates.
- Heat the lysates for 5-10 minutes at 100C.
- Filter the buffers before use.
- Block the membrane with 3-5% BSA in all cases for 2 hours at room temperature.
- Please make sure the lysates are prepared in ice cold RIPA buffer. Keep the lysates on ice.
- Use TBST as dilution and washing buffer.
- Try loading control with same lysates and no primary control (no primary antibody)>
- Please check the cells should be 80-90% confluent.
- use fresh protease inhibitors.
I hope these suggestions would help to improve results. I will look forward to hearing from you soon.

Read More

Question

This email has 2 sections:

A recap of the complaint

The respondent’s survey results (section-by-section)
Recap of the complaint

Date(s) submitted: July 9, 2012

Catalog number(s): 13915

Product(s): Mouse monoclonal [40C1285] to TLR3

CCEID: 3934045
Survey results
Telephone section
Q. Did you contact Abcam's Scientific Support by telephone in reference to this complaint?
A. No
Q. Was it clear from the phone options which number you needed to press to get your issue resolved?
A.
Protocol advice section
Q. Did Abcam's Scientific Support give you protocol advice?
A.Yes
Q. How did you feel about being given protocol advice?
A.I requested protocol advice
Q. Did the product work successfully after following the advice?
A.No
Q. Why have you not tried the protocol advice? (Select all that apply.)
Free of charge replacement section
Q. Did Abcam's Scientific Support give you a free of charge replacement?
A. No

Q. Did the replacement product work successfully?
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.Satisfied
Q. Could we have done anything to better resolve the problem?
A.dont know!
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.
A.WE found ourself a solution for the problem and got satisfying staining with the product.

Read More
Answer

Thank you for completing the survey.

I am glad to hear that the product worked fine at the end. Could you share the details of step you have altered for better results; it may help other scientists in community?

Many thanks for your cooperation during the whole case.

Best of luck with you research.

Read More

Answer

Thank you for your email. I am sorry to hear that you have been experiencing problems with this antibody.

The protocol seems fine to me however because antibodies needs optimization when they used with paraffin embedded sections so I have following suggestions, that might help to improve results;

- Recommend trying antigen retrieval with microwave (700 watt full power) 5-10 minutes incubation in citrate buffer pH 6.0. or EDTA buffer pH 9.0. Please carefully check the buffer pH, in many cases the incorrect pH fails the antibodies.
- Pressure cooker; the slides can be cooked for 2 minutes at full required pressure.
- Could you provide the order number?

I am sure optimizing the antigen retrieval step would help in getting good staining. However, if the results do not improve please contact me for further assistance.

Read More

Answer

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.


I look forward to receiving your reply.

Read More

Answer

Thank you for contacting us.

We do notsell blocking peptide for ab12121 so I am sorry we will not be able to provide this.

I have been waiting an answer from lab regarding ab13915 and ab45371; I will send you the info soon.

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

Read More

Answer

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for one vial of ab13915.

To check the status of the order please contact our Customer Service team and reference this number.
Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.
I wish you the best of luck with your research.

Read More

1-10 of 12 Abreviews or Q&A

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

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