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. The antibody recognizes mouse CCR4 and has no cross-reactivity to human CCR4.
  • Tested applications
    Suitable for: IHC-Fr, ELISA, ICC, WB, IHC-P, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse
    Does not react with: Human
  • Immunogen

    Synthetic peptide:

    DTTQDETVYNSYYFYESMPC

    , corresponding to amino acids 8-26 of Mouse CCR4.

  • Positive control
    • Mouse spleen cells

Properties

Applications

Our Abpromise guarantee covers the use of ab1664 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-Fr Use at an assay dependent concentration.
ELISA 1/500000.
ICC 1/500.
WB 1/1000.
IHC-P Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

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

 

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

References

This product has been referenced in:
  • Gülden E  et al. Microbiota control immune regulation in humanized mice. JCI Insight 2:N/A (2017). Mouse . Read more (PubMed: 29093268) »
  • Richter JR  et al. Macrophage-derived chemokine (CCL22) is a novel mediator of lung inflammation following hemorrhage and resuscitation. Shock 42:525-31 (2014). IHC-P ; Mouse . Read more (PubMed: 25136780) »
See all 14 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question

Thanks for your reply. Please see responses below. Apologies, but I don't have the PO number immediately available. The lot number is 48073. 1. Please describe the problem (high background, low signal, no signal etc). There does appear to be high background with the combination of ab1664, the ab6740 secondary, and a strepavidin-fluorophore from Pharmingen. I've tried strepavidin-PE, APC, TexasRed, etc. 2. On what material are you testing the antibody in FACS? •Species? •Cell type? •Cell line? I've been looking at epidermal cells from B6 mouse ear, gating on scatter characteristics, then CD45+ CD3+ (T-cells), and looking for CCR4 positive cells. 3. Which buffer did you use for cell suspension? We stain in FACS buffer, which is normal PBS + 2% FCS + 0.05% sodium azide. 4. How many cells did you use? ~1 million epidermal cells per sample. 5. Did you permeabilize the cells? How? No. 6. Primary antibody •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)? Dilution is 1 microliter of ab1664 per 50 microliters (the volume we stain in). We incubate for ~15 to 30 minutes. We then wash with FACS buffer (PBS + sodium azide + FCS), and then repeat the staining procedure with the secondary rabbit antibody and strepavidin+fluorophore tertiary. 7. Secondary antibody •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? ab6740, also dilution of 1 microliter of ab6740 per 50 microliters for a stain of one million cells, as above. Washing is as above. I then apply the strepavidin-fluorophore at the same dilution and incubate (again usually about 15 minutes). This is followed by a final wash. We usually FACS 1 million cells in a final volume of 200 microliters. 8. Which detection system did you use? •Detection wavelength The machine is a BD biosciences LSR 1. I've used a variety of fluorophores, corresponding to multiple wavelengths for FITC, PE, TexasRed, APC, and APC/Cy7. 9. Did you apply positive and negative controls along with the samples? Please specify. Negative controls has been splenocytes from normal B6 mice, which are expected to have cells that largely lack mCCR4. No positive controls have been really available. The strepavidin-fluorophores are known to work as they're widely used with a number of other biotinylated antibodies (admittedly many from Pharmingen). The rabbit anti goat secondary hssn't been tested because the only other goat antibody we have is ab1661 (anti-CCR10 and not advertised by Abcam as suitable for FACS. Been having the same issues with that one as well, but that's a different story). 10. Optimization attempts •How many times have you tried the FACS? •Do you obtain the same results every time? •What steps have you altered? Have tried the FACS 3 or 4 times. I've maintainly altered procedures for the collection of epidermal cells, but haven't really tried changing the FACS staining or acquisition steps.

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Answer

Thank you very much for your patience and for the details in which you have provided. I contacted the originator of ab1664 and below is the FACS protocl that they used. I hope this will be of use to you and if you continue to experience difficulty with this antibody please let me know and I will send you a replacement or a refund. FACS Protocol: 1. Pellet approx. 200,000 cells by centrifugation. 2. Resuspend the cell pellet in 50-75ul of medium with 5% FBS. 3. Add 1-5 ul of the anti-CCR antibody. 4. Incubate at 4 degree C for 45 minutes. 5. Wash the cells two or more times before adding secondary antibody. 6. Try using FITC conjugated F(ab’)2 anti goat antibody at appropriate dilution. If using anti-goat IgG then a blocking step indicated below is strongly recommended to reduce non-specific background. 7. Wash the cells and analyze. Controls and Blocking Steps: 1. Blocking of Fc-receptors: If one intends to use secondary IgG antibody raised in rabbits then the investigator initially should pre-incubate mouse cells with normal rabbit IgG or 2% heat inactivated normal rabbit serum for 30 minutes and use secondary rabbit antibody system at appropriate dilution with 100ug/ml of normal rabbit IgG. 2. Negative Control: Use another antigen affinity purified goat antibody at similar concentration to a protein that is not expressed on mouse cells as negative control.

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Answer

Thank you very much for your enquiry and patience. At this time, the only publication that we are currently aware of featuring the use of ab1664 is the Hoogewerf A et al. reference. We also don't have any images/figures to share at this time. If you can provide me with some more information regarding your protocol (please see the questions below), we can help you out. Thank you, and I look forward to hearing from you. I have some general questions, the answers to which will enable me to investigate this matter as quickly as possible. Also, please include the antibody lot number (located on the vial) and the Abcam order number or purchase order number that was used. 1. Please describe the problem (high background, low signal, no signal etc). 2. On what material are you testing the antibody in FACS? •Species? •Cell type? •Cell line? 3. Which buffer did you use for cell suspension? 4. How many cells did you use? 5. Did you permeabilize the cells? How? 6. Primary antibody •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 •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. Which detection system did you use? •Detection wavelength 9. Did you apply positive and negative controls along with the samples? Please specify. 10. Optimization attempts •How many times have you tried the FACS? •Do you obtain the same results every time? •What steps have you altered?

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Answer

Cysteine molecules are commonly placed on the end of an immunising amino acid sequence. The reason for this is two fold: 1) It allows the peptide to be sulphur-linked to a suitable carrier molecule to provoke an immune response in the host animal. 2) It allows the peptide to be sulphur-linked to a suitable column for affinity purification of the resulting antibody. This is due to cysteine being the only amino acid to possess an SH- group. This extras cysteine will not affect the performance of the antibody. If you have any more questions then please don't hesitate to ask.

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