Overview

  • Product name
  • Description
    Goat polyclonal to CCR4
  • Host species
    Goat
  • Specificity
    Peptide sequence is < 50 % identical to other human chemokine receptors in this region.
  • Tested applications
    Suitable for: Flow Cyt, ICC/IF, ELISA, ICC, IHC-P, WB, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Human, Cynomolgus monkey
  • Immunogen

    Synthetic peptide:

    SNYYLYESIPKPCTKEGIKAFGE

    , corresponding to amino acids 17-39 of Human CCR4 (extracellular domain).

  • Positive control
    • Human peripheral blood cells or paraffin sections of spleen. IHC-P:FFPE mouse lymph node normal. IHC-P:FFPE human tonsil normal. IHC-P:FFPE rat spleen normal.

Properties

Applications

Our Abpromise guarantee covers the use of ab1669 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 Use at an assay dependent concentration.

ab37373 - Goat polyclonal IgG, is suitable for use as an isotype control with this antibody.

 

ICC/IF Use at an assay dependent concentration. Fix cells with 4% PFA (see Ritter et al).
ELISA 1/100000.
ICC 1/300.
IHC-P 1/300.
WB 1/1000. Detects a band of approximately 55 kDa (predicted molecular weight: 42 kDa).
IHC-Fr Use at an assay dependent concentration. Fix in acetone at -20°C for 5 minutes. See Heller et al.

Target

  • Function
    High affinity receptor for the C-C type chemokines CCL17/TARC and CCL22/MDC. The activity of this receptor is mediated by G(i) proteins which activate a phosphatidylinositol-calcium second messenger system. Can function as a chemoattractant homing receptor on circulating memory lymphocytes and as a coreceptor for some primary HIV-2 isolates. In the CNS, could mediate hippocampal-neuron survival.
  • Tissue specificity
    Predominantly expressed in the thymus, in peripheral blood leukocytes, including T-cells, mostly CD4+ cells, and basophils, and in platelets; at lower levels, in the spleen and in monocytes. Detected also in macrophages, IL-2-activated natural killer cells and skin-homing memory T-cells, mostly the ones expressing the cutaneous lymphocyte antigen (CLA). Expressed in brain microvascular and coronary artery endothelial cells.
  • Sequence similarities
    Belongs to the G-protein coupled receptor 1 family.
  • Post-translational
    modifications
    In natural killer cells, CCL22 binding induces phosphorylation on yet undefined Ser/Thr residues, most probably by beta-adrenergic receptor kinases 1 and 2.
  • Cellular localization
    Cell membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • C C chemokine receptor type 4 antibody
    • C C CKR 4 antibody
    • C-C chemokine receptor type 4 antibody
    • C-C CKR-4 antibody
    • CC CKR 4 antibody
    • CC-CKR-4 antibody
    • CCR 4 antibody
    • CCR-4 antibody
    • CCR4 antibody
    • CCR4_HUMAN antibody
    • CD194 antibody
    • Chemokine (CC motif) receptor 4 antibody
    • chemokine C C motif receptor 4 antibody
    • ChemR13 antibody
    • CKR4 antibody
    • CMKBR 4 antibody
    • CMKBR4 antibody
    • HGCN 14099 antibody
    • K5 5 antibody
    • K5-5 antibody
    • MGC88293 antibody
    see all

Images

  • ab1669 staining CCR4 in Mouse whole blood by Flow Cytometry. Red blood cells were lysed in PBS + 1% BSA and 0.01% sodium azide and fixed in paraformaldehyde. The sample was incubated with the primary antibody (1/100 in PBS + 1% BSA and 0.01% sodium azide) for 1 hour at 4°C. A biotin-conjugated Donkey anti-goat IgG polyclonal (1/200) was used as the secondary antibody.
    Gating Strategy: Live Lymphocytes.

    See Abreview

  • IHC image of CD3 staining in a formalin fixed, paraffin embedded normal rat spleen tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab1669 at 1/100 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

     

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

     

  • Frozen sections of  human skin tissue stained for CCR4 using ab1669 in immunohistochemical analysis (green). CD31(red) and nuclei (blue) are also shown. The right hand panel is a merged image.

    Image courtesy of PMID 25915746 (PLoS One 2015 Apr 27;10(4)).

  • IHC image of CD3 staining in a formalin fixed, paraffin embedded normal human tonsil tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab1669 at 1/100 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

     

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • IHC image of CD3 staining in TISSUE formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab1669 at 1/100 dilution for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

     

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • Immunohistochemistry using ab1669 on a section of human spleen.

  • IHC image of CCR4 staining in human spleen formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab1669, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
  • Anti-CCR4 antibody (ab1669) at 1 µg/ml + Human thymus tissue lysate - total protein (ab30146) at 10 µg

    Secondary
    Rabbit polyclonal to Goat IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 42 kDa
    Observed band size: 55 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 100 kDa, 37 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 12 minutes


    CCR4 contains a number of potential glycosylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.

References

This product has been referenced in:
  • Maolake A  et al. Tumor-associated macrophages promote prostate cancer migration through activation of the CCL22-CCR4 axis. Oncotarget 8:9739-9751 (2017). Human . Read more (PubMed: 28039457) »
  • Berlato C  et al. A CCR4 antagonist reverses the tumor-promoting microenvironment of renal cancer. J Clin Invest 127:801-813 (2017). Read more (PubMed: 28134623) »
See all 10 Publications for this product

Customer reviews and Q&As

1-10 of 11 Abreviews or Q&A

Answer

Merci beaucoup pour toutes ces informations détailléesqui nous sont très utiles pour identifier l'origine du problème rencontré avec le ab1669 lot GR34771-3.

Je n'ai rien à modifier à votre protocole qui me semble être optimal. La quantité de protéines (30µg) est ce que nous recommandons, le test de différentes dilutions d'anticorps primaire ainsi que l'utilisation de différents agents bloquants (lait ou BSA) sont également essentiels; parfait!

Nous n'avons malheureusement pas encore trouvé de publication faisant référence à l'anticorps ab113085. Quant au peptide utilisé comme immunogène, je vais demander au laboratoire produisant cet anticorps si la séquence peut être communiquée.

Je vous contacterai de nouveau dès que j'aurai reçu une réponse.

Merci pour votre patience et n'hésitez pas à m'informer des résultats obtenus avec l'anticorps de remplacement ab113085.

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Answer

Merci de nous avoir contactés.

Nous sommes désolés d'apprendre que le produit ab1669 que vous avez reçu ne fonctionne pas comme attendu.

Nos produits sont garantis pour fonctionner tels qu'ils sont décrits sur la fiche technique, nous pouvons donc effectivement résoudre le problème que vous avez rencontré en vous envoyant un anticorps de remplacement.

Les 2 anticorps ab83250et ab113085 (comme ab1669) sont garantis pour fonctionner en Western Blot et sur échantillons humains.
L'anti-CCR4 ab83250 n'étant pas actuellement en stock, j'ai mis en place l'envoi d'une unité de ab113085. Le numéro de commande de remplacement gratuit de ce produit est *******. Vous recevrez prochainement un mail de confirmation comprenant les détails d'expédition.

En échange, nous serions ravis si vous pouviez nous communiquer quelques détails concernant votre protocole en remplissant le questionnaire technique ci-joint. Ces informations sont très importantes pour nous car elles nous permettent d'investiguer la source du problème rencontré avec cet anticorps et de prendre les dispositions nécessaires pour assurer une bonne qualité de nos produits.

N'hésitez pas à nous contacter lors d'une prochaine occasion.

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Application
Flow Cytometry
Sample
Cynomolgus Monkey Cell (Whole blood)
Specification
Whole blood
Preparation
Cell harvesting/tissue preparation method: After red blood cell lysis
Sample buffer: PBS/BSA 1%/Azide 0.01%
Fixation
Paraformaldehyde
Permeabilization
No
Gating Strategy
alive lymphocytes

Abcam user community

Verified customer

Submitted Apr 27 2012

Abcam has not validated the combination of species/application used in this Abreview.
Application
Flow Cytometry
Sample
Rat Cell (Splenocytes)
Specification
Splenocytes
Preparation
Cell harvesting/tissue preparation method: spleen was mashed
Sample buffer: PBS/BSA 1%/Azide 0.01%
Permeabilization
No
Gating Strategy
alive lymphocytes

Abcam user community

Verified customer

Submitted Apr 18 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Flow Cytometry
Sample
Mouse Cell (Whole blood)
Specification
Whole blood
Preparation
Cell harvesting/tissue preparation method: After red blood cell lysis
Sample buffer: PBS/BSA 1%/Azide 0.01%
Fixation
Paraformaldehyde
Permeabilization
No
Gating Strategy
alive lymphocytes

Abcam user community

Verified customer

Submitted Apr 18 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Flow Cytometry
Sample
Human Cell (PBMC)
Specification
PBMC
Preparation
Cell harvesting/tissue preparation method: after ficoll
Sample buffer: PBS/BSA 1%/ Azide 0.01%
Permeabilization
No
Gating Strategy
on monocytes

Abcam user community

Verified customer

Submitted Dec 28 2011

Answer

Thank you for your enquiry. Ab1667: Antigen Retrieval: Sections were treated with antigen retrieval solution (Dako # S-1700, pH 6.0) and steamed for 20 minutes followed by cooling for 20 minutes. Variations of the method will work successfully as shown in the following publication that has images as well: http://clincancerres.aacrjournals.org/cgi/content/full/11/7/2459 Ab1669: Antigen Retrieval: Sections were treated with antigen retrieval solution (Dako # S-1700, pH 6.0 or #S-3308, pH 8.0) and steamed for 20 minutes followed by cooling for 20 minutes. I hope that this information helps. Should you have any further questions then please do not hesitate to get back in touch with me.

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Answer

Thank you for your patience. The originator of ab12953 and ab13396 has gotten back to me with the standard IHC protocol that they follow which I have included below. If you have any additional questions, please let me know. Tissue Preparation: Formalin fixation and embedding in paraffin wax Tissue Sectioning: Make 4-µm sections and place on pre-cleaned and charged microscope slides. Heat in a tissue-drying oven for 45 minutes at 60°C. Deparaffinization: Wash dry slides in 3 changes of xylene – 5 minutes each @ RT Rehydration: Wash slides in 3 changes of 100% alcohol – 3 minutes each @ RT Wash slides in 2 changes of 95% alcohol – 3 minutes each @ RT Wash slides in 1 change of 80% alcohol – 3 minutes @ RT Rinse slides in gentle running distilled water – 5 minutes @ RT Antigen retrieval: Steam slides in 0.01 M sodium citrate buffer, pH 6.0 at 99-100°C - 20 minutes Remove from heat and let stand at room temperature in buffer - 20 minutes Rinse in 1X TBS with Tween (TBST) – 1 minute @ RT Immunostaining: (Do not allow tissues to dry at any time during the staining procedure) Apply a universal protein block – 20 minutes @ RT Drain protein block from slides, apply diluted primary antibody – 45 minutes @ RT Rinse slides in 1X TBST - 1 minute @ RT Apply a biotinylated anti-rabbit IgG (H+L) secondary – 30 minutes @ RT Rinse slides in 1X TBST - 1 minute @ RT Apply alkaline phosphatase streptavidin – 30 minutes @ RT Rinse slides in 1X TBST - 1 minute @ RT Apply alkaline phosphatase chromogen substrate – 30 minutes @ RT Wash slides in distilled water – 1 minute @ RT Dehydrate: (This method should only be used if the chromogen substrate is alcohol insoluble (e.g. Vector Red, DAB) Wash slides in 2 changes of 80% alcohol – 1 minute each @ RT Wash slides in 2 changes of 95% alcohol – 1 minute each @ RT Wash slides in 3 changes of 100% alcohol – 1 minute each @ RT Wash slides in 3 changes of xylene – 1 minute each @ RT Apply coverslip

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Answer

Thank you for your phone call. For ab13268, ab1662, and ab1669, the originator has informed me that the antigen retrieval method for IHC-formalin fixed, paraffin embedded sections involved treating the sections with antigen retrieval solution (Dako # S-1700, pH 6.0 or #S-3308, pH 8.0) and steamed for 20 minutes followed by cooling for 20 minutes. I will contact you again when I receive information regarding ab12953 and ab13396. Have a great weekend!

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Question

I will try to answer your questions so you can help me. We have an answer: 1. Please describe the problem (high background, no staining etc). We found high background staining in all kryostat sections independent of the concentration of the primary antibody. 2. On what material are you testing the antibody in IHC? We made a positive control by injection of CCR4 positive CEM-cells (human leucaemia T-cell line) into human healthy skin. Then we made kryostat sections (6 µm). * Species? * Cell line? * Tissue? 3. How did you fix the samples? We fixed the samples before staining with acetone for 10 minutes. * Ethanol, methanol * Acetone * Paraformaldehyde * Other 4. Did you apply antigen retrieval step? We used a biotinylated secondary antibody, then ABC-kit (Vectastain). The substrate was DAB solution * Enzymatic method * Heat mediated technique * Other 5. How did you block the unspecific binding sites? We quenched endogenous peroxidases with 1% H2O2 (40 min). We blocked unspecific bindings with 1% bovine serum albumin (RT, humid chamber, 30 min). 6. Primary antibody The CCR4 antibody was a goat anti human CCR4 antibody. Dilutions: as descibed 1:300, we also tried 1:50, 1:150, 1:100, 1:500. We incubated the primary antibody overnight at 4° in a humid chamber. We washed in three steps with high volume (3 times a 10 min.) * Specification (in which species was it raised against)? * At what dilution(s) have you tested this antibody? * Incubation time, wash steps (multiple short washes are more effective than fewer longer wash steps)? 7. Secondary antibody We used a biotinylated anti-goat IgG antibody (produced in rabbit, Fa. Vector) Dilution 1:200, Incubation time 45 min. at RT, washing with PBS 2x10 min., 45 min. ABC reagent in humid chamber at RT, washing 2x10 min with PBS, 5 min. DAB solution. * What secondary antibody are you using? * Specification (in which species was it raised against)? * At what dilution(s) have you tested this antibody? * Incubation, wash steps? * Do you know whether the problems you are experiencing come from the secondary? * What detection method are you using? 8. Background staining See images of the attachment. "über" means overview. We here used the described antibody dilution 1:300. Only in this dilution we saw marked brown cells (CEM-cells). "pat1" means we stained skin from a patient with atopic dermatitis. * Please provide an image of your staining 9. Which detection system did you use? ABC-kit, Vaectastain 10. Did you apply positive and negative controls along with the samples? Please specify. yes, "patneg" means negative control (without primary antibody) Please see attachment. 11. Optimization attempts only a few times, we try to establish the protocol. We altered the washing steps (higher volumes of PBS), we tried different dilutions. We always saw high background staining. * How many times have you tried the IHC? * Do you obtain the same results every time? * What steps have you altered? We look forward to hearing from you soon.

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Answer

Thank you for submitting the completed questionnaire to us and for your patience. We are very sorry to hear that you are having problem with this antibody. We have searched our database and found that this is a popular selling product and your feedback is the first we have received about it not working. Therefore, at this stage, we would suggest that there is either a problem with the vial you received, or modifications to your protocol are needed to obtain a positive result. As we understand from your e-mails, you performed IHC on skin biopsies taken from patients with atopic dermatitis. We would strongly suggest using positive control along with the samples. Human spleen sections or human peripheral blood cells would be good for this purpose. This is especially important since there are some publications which suggest that expression of CCR4 in skin-derived lymphocytes is higher that its expression in other type of tissues. Lymphocytes from inflamed skin cells have especially high levels of CCR4 which may be one of the reasons why you are having such a high background signal. Please take a look at this published paper; you may find some useful information: http://ajp.amjpathol.org/cgi/content/full/160/1/347 You may also need to alter certain steps in your protocol. Try to increase the concentration of the blocking serum i.e. 3-5% BSA to see if the background signal is getting lower. Block the endogenous peroxidase activity with 1.6% (not 1%) H2O2, it may help. We have looked at the images you have forwarded to us, it is clear that the antibody works. Comparing the intensity of the signal in the negative control and in the samples, the staining is definitely stronger in the biopsy samples. Although, it is true that the background signal in some areas of the sections is a bit high. We hope this will be useful for you. Should you still have any problem, then please do not hesitate to contact us again.

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1-10 of 11 Abreviews or Q&A

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