Overview

  • Product name

    Anti-TLR9 antibody [26C593.2]
    See all TLR9 primary antibodies
  • Description

    Mouse monoclonal [26C593.2] to TLR9
  • Host species

    Mouse
  • Tested applications

    Suitable for: WB, IHC-Fr, Flow Cyt, ICC/IF, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Horse, Dog, Human, Rhesus monkey
    Predicted to work with: Chimpanzee, Cynomolgus monkey, Gorilla, Orangutan
  • Immunogen

    Synthetic peptide corresponding to Human TLR9 aa 268-284 conjugated to keyhole limpet haemocyanin. AAF78037
    Sequence:

    CPRHFPQLHPDTFSHLS


    Database link: Q9NR96

  • Positive control

    • WB: Human peripheral blood mononuclear cells (PBMCs). Human, mouse and rat intestine tissue lysates. Flow Cytometry: Ramos and PBMC cells. IHC-Fr: Human lung adenocarcinoma and malignant lung tissues. IHC-P: Human spleen tissue. ICC/IF: A549 cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab134368 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
WB Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 120 kDa (predicted molecular weight: 115 kDa).
IHC-Fr Use a concentration of 10 - 20 µg/ml.
Flow Cyt Use 0.1-2µg for 106 cells.

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

 

ICC/IF Use at an assay dependent concentration.
IHC-P Use a concentration of 5 µg/ml.

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. TLR9 is a nucleotide-sensing TLR which is activated by unmethylated cytidine-phosphate-guanosine (CpG) dinucleotides. Acts via MYD88 and TRAF6, leading to NF-kappa-B activation, cytokine secretion and the inflammatory response.
  • Tissue specificity

    Highly expressed in spleen, lymph node, tonsil and peripheral blood leukocytes, especially in plasmacytoid pre-dendritic cells. Levels are much lower in monocytes and CD11c+ immature dendritic cells. Also detected in lung and liver.
  • Sequence similarities

    Belongs to the Toll-like receptor family.
    Contains 26 LRR (leucine-rich) repeats.
    Contains 1 TIR domain.
  • Cellular localization

    Endoplasmic reticulum membrane. Endosome. Lysosome. Cytoplasmic vesicle > phagosome. Relocalizes from endoplasmic reticulum to endosome and lysosome upon stimulation with agonist.
  • Information by UniProt
  • Database links

  • Alternative names

    • CD 289 antibody
    • CD289 antibody
    • TLR 9 antibody
    • TLR9 antibody
    • TLR9_HUMAN antibody
    • Toll like receptor 9 antibody
    • Toll like receptor 9 isoform A precursor antibody
    • Toll like receptor 9 isoform B antibody
    • Toll-like receptor 9 antibody
    see all

Images

  • Flow Cytometric analysis of TLR9 in Ramos (Human Burkitt's lymphoma cell line) cells labelled with ab134368 at 0.1 µg (pink), no antibody (blue) or isotype control antibody (green). Goat anti-mouse IgG FITC secondary was used.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human spleen tissue labelling TLR9 with ab134368 at 5 µg/ml.

    Staining was enhanced by boiling tissue sections in 10 mM sodium citrate buffer, pH 6.0 for 10-20 minutes followed by cooling at room temperature for 20 minutes.

  • All lanes : Anti-TLR9 antibody [26C593.2] (ab134368) at 3 µg/ml

    Lane 1 : Human peripheral blood mononuclear cells (PBMCs)
    Lane 2 : Human intestine tissue lysate
    Lane 3 : Mouse intestine tissue lysate
    Lane 4 : Rat intestine tissue lysate

    Lysates/proteins at 40 µg per lane.

    Secondary
    All lanes : Goat anti-mouse HRP conjugate

    Predicted band size: 115 kDa
    Observed band size: 120 kDa
    why is the actual band size different from the predicted?

  • Flow Cytometric analysis of TLR9 in human PBMC cells labelled with ab134368 at 0.5 µg (red) or an isotype control antibody (green).

  • Immunohistochemical analysis of TLR9 in frozen sections of human lung adenocarcinoma tissue, using ab134368 at a 1/100 dilution.

  • Immunohistochemical analysis of TLR9 in frozen sections of A549 (Human lung carcinoma cell line) cells, using ab134368 at a 1/100 dilution.

  • Immunohistochemical analysis of TLR9 in frozen sections of malignant human lung tissue, using ab134368 at a 1/100 dilution.

References

This product has been referenced in:

  • Guo HY & Cui ZJ Extracellular Histones Activate Plasma Membrane Toll-Like Receptor 9 to Trigger Calcium Oscillations in Rat Pancreatic Acinar Tumor Cell AR4-2J. Cells 8:N/A (2018). Read more (PubMed: 30577532) »
  • Barboza R  et al. TLR4-Mediated Placental Pathology and Pregnancy Outcome in Experimental Malaria. Sci Rep 7:8623 (2017). IHC-P ; Mouse . Read more (PubMed: 28819109) »
See all 4 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

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 with the order number xxx with ab52967. The estimated delivery date is xxx. I apologize about this delay.

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.

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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,

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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.

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Answer

Thank you for contacting us.

My colleague got back to me and we have now re-published ab52967.
Could you please just quickly confirm that I will send you 1 vial of ab52967 free of charge to replace ab134368?
The estimated delivery date will be in about 1-2 weeks, but I will have a more concrete date, once I create the free of charge order and will let you know it.

I look forward to hear back from you hope and resolve this case.

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

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Answer

Thank you for contacting us.

From the lab which producess the ab134368 Anti-TLR9 antibody [26C593.2]I got the following reply:

"Clone 26C593.2 and 26C593 is the same antibody, with and without the subclone designation. The antigen information is listed on the data sheet: Antigen
Clone 26C593.2 and 26C593 is the same antibody, with and without the subclone designation. This antibody was developed against KLH-conjugated synthetic peptide corresponding to amino acids 268-284 (CPRHFPQLHPDTFSHLS) of human TLR9 isoform A (Genbank accession no. AAF78037)."

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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