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    ccr4-antibody-ab1669.pdf

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Immunology Innate Immunity Chemokines Beta Chemokine Rec. (CCR)
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Anti-CCR4 antibody (ab1669)

  • Datasheet
  • SDS
Reviews (4)Q&A (7)References (12)

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Abpromise

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Western blot - Anti-CCR4 antibody (ab1669)

    Key features and details

    • Goat polyclonal to CCR4
    • Suitable for: WB
    • Reacts with: Human, Cynomolgus monkey
    • Isotype: IgG

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    Overview

    • Product name

      Anti-CCR4 antibody
      See all CCR4 primary antibodies
    • Description

      Goat polyclonal to CCR4
    • Host species

      Goat
    • Specificity

      Peptide sequence is < 50 % identical to other human chemokine receptors in this region.
    • Tested Applications & Species

      Application Species
      WB
      Human
      See all applications and species data
    • Immunogen

      Synthetic peptide:

      SNYYLYESIPKPCTKEGIKAFGE

      , corresponding to amino acids 17-39 of Human CCR4 (extracellular domain).
      Run BLAST with BLAST the sequence with ExPASy Run BLAST with BLAST the sequence with NCBI

    Properties

    • Form

      Liquid
    • Storage instructions

      Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
    • Storage buffer

      Preservative: 0.1% Sodium azide
      Constituent: 0.1% BSA
    • Concentration information loading...
    • Purity

      Immunogen affinity purified
    • Clonality

      Polyclonal
    • Isotype

      IgG
    • Research areas

      • Immunology
      • Innate Immunity
      • Chemokines
      • Beta Chemokine Rec. (CCR)
      • Cardiovascular
      • Atherosclerosis
      • Vascular Inflammation
      • Leukocyte recruitment
      • Chemokines

    Associated products

    • Compatible Secondaries

      • Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) (ab150129)
      • Donkey Anti-Goat IgG H&L (HRP) (ab205723)
    • Conjugation kits

      • PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
      • APC Conjugation Kit - Lightning-Link® (ab201807)
    • Isotype control

      • Goat IgG, polyclonal - Isotype Control (ab37373)
    • Recombinant Protein

      • Recombinant Human CCR4 protein (ab253173)

    Applications

    The Abpromise guarantee

    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.

    Guaranteed

    Tested applications are guaranteed to work and covered by our Abpromise guarantee.

    Predicted

    Predicted to work for this combination of applications and species but not guaranteed.

    Incompatible

    Does not work for this combination of applications and species.

    Application Species
    WB
    Human
    Application Abreviews Notes
    WB
    1/1000. Detects a band of approximately 55 kDa (predicted molecular weight: 42 kDa).
    Notes
    WB
    1/1000. Detects a band of approximately 55 kDa (predicted molecular weight: 42 kDa).

    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.
    • Target information above from: UniProt accession P51679 The UniProt Consortium
      The Universal Protein Resource (UniProt) in 2010
      Nucleic Acids Res. 38:D142-D148 (2010) .

      Information by UniProt
    • Database links

      • Entrez Gene: 1233 Human
      • GenBank: NP_005499.1 Human
      • Omim: 604836 Human
      • SwissProt: P51679 Human
      • Unigene: 184926 Human
      • 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

      • Western blot - Anti-CCR4 antibody (ab1669)
        Western blot - Anti-CCR4 antibody (ab1669)
        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.

      Protocols

      • Western blot protocols

      Click here to view the general protocols

      Datasheets and documents

      • Datasheet
      • SDS
    • References (12)

      Publishing research using ab1669? Please let us know so that we can cite the reference in this datasheet.

      ab1669 has been referenced in 12 publications.

      • Yuan FY  et al. Tanshinone IIA improves diabetes mellitus via the NF-?B-induced AMPK signal pathway. Exp Ther Med 16:4225-4231 (2018). PubMed: 30344697
      • Wang Y  et al. HIC1 deletion promotes breast cancer progression by activating tumor cell/fibroblast crosstalk. J Clin Invest 128:5235-5250 (2018). PubMed: 30204129
      • Berlato C  et al. A CCR4 antagonist reverses the tumor-promoting microenvironment of renal cancer. J Clin Invest 127:801-813 (2017). PubMed: 28134623
      • Maolake A  et al. Tumor-associated macrophages promote prostate cancer migration through activation of the CCL22-CCR4 axis. Oncotarget 8:9739-9751 (2017). Human . PubMed: 28039457
      • Ou B  et al. CCR4 promotes metastasis via ERK/NF-?B/MMP13 pathway and acts downstream of TNF-a in colorectal cancer. Oncotarget 7:47637-47649 (2016). IHC . PubMed: 27356745
      View all Publications for this product

      Customer reviews and Q&As

      Show All Reviews Q&A
      Submit a review Submit a question

      1-10 of 11 Abreviews or Q&A

      Question

      Bonjour,
      Merci de votre rapidité de réponse.
      Je vous retourne en échange votre document complété et mes images de blots.
      N'hésitez pas à me faire des remarques sur ceux ci pour augmenter mes chances de réussite avec le nouvel Ac.
      Dans la fiche technique de l'Ac que vous me faites parvenir (ab113085), le peptide ciblé (séquence, localisation sur la protéine) n'est pas précisé, le connaissez vous?
      Avez vous des publications référencées avec cet Ac car j'avais choisi l'ab1669 pour cela?
      Cordialement,

      Read More

      Abcam community

      Verified customer

      Asked on Sep 06 2012

      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.

      Read More

      Abcam Scientific Support

      Answered on Sep 06 2012

      Question


      Nous vous avons acheté un Ac fin avril et après de multiples essais celui ci ne
      révèle pas la protéine souhaitée (+ beaucoup de non spécifique):
      Anticorps anti-CCR4 (ab1669)
      Cellules testées = cellules humaines
      Application WB
      Pouvez vous me dire qu'elles sont les modalités / conditions pour remplacer cet
      Ac par un autre?
      Je serais éventuellement intéressée pour tester soit l'ab83250 ou l'ab113085
      selon vos conseils.
      Cordialement,

      Read More

      Abcam community

      Verified customer

      Asked on Sep 04 2012

      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.

      Read More

      Abcam Scientific Support

      Answered on Sep 04 2012

      Flow Cytometry abreview for Anti-CCR4 antibody

      Poor
      Abreviews
      Abreviews
      abreview image
      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
      Read More

      Abcam user community

      Verified customer

      Submitted Apr 27 2012

      Flow Cytometry abreview for Anti-CCR4 antibody

      Poor
      Abreviews
      Abreviews
      abreview image
      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
      Read More

      Abcam user community

      Verified customer

      Submitted Apr 18 2012

      Flow Cytometry abreview for Anti-CCR4 antibody

      Good
      Abreviews
      Abreviews
      abreview image
      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
      Read More

      Abcam user community

      Verified customer

      Submitted Apr 18 2012

      Flow Cytometry abreview for Anti-CCR4 antibody

      Average
      Abreviews
      Abreviews
      abreview image
      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
      Read More

      Abcam user community

      Verified customer

      Submitted Dec 28 2011

      Question

      I have a customer who purchased your antibodies, ab1669 and ab1667. The customer would like to use them for immunohistochemistry of paraffin-embedded sections. Her sample is human lymphoid neoplasm. Please let me know an antigen retrieval method you recommend, respectively. And regarding each figure of IHC-formalin fixed, paraffin embedded sections please let me know also each antigen retrieval method when you got these data.

      Read More

      Abcam community

      Verified customer

      Asked on Feb 07 2007

      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.

      Read More

      Abcam Scientific Support

      Answered on Feb 08 2007

      Question

      For each of these antibodies, specifically what type of antigen retrieval was used for IHC-formalin fixed, paraffin embedded sections?

      Read More

      Abcam community

      Verified customer

      Asked on Jan 07 2005

      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

      Read More

      Abcam Scientific Support

      Answered on Jan 12 2005

      Question

      For each of these antibodies, specifically what type of antigen retrieval was used for IHC-formalin fixed, paraffin embedded sections?

      Read More

      Abcam community

      Verified customer

      Asked on Jan 07 2005

      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!

      Read More

      Abcam Scientific Support

      Answered on Jan 07 2005

      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.

      Read More

      Abcam community

      Verified customer

      Asked on Dec 15 2004

      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.

      Read More

      Abcam Scientific Support

      Answered on Dec 17 2004

      1-10 of 11 Abreviews or Q&A

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