Overview

  • Product name
    Anti-CXCR4 antibody [UMB2]
    See all CXCR4 primary antibodies
  • Description
    Rabbit monoclonal [UMB2] to CXCR4
  • Host species
    Rabbit
  • Specificity
    Although some customers can get this ab to work in mouse and rat successfully we cannot reproduce this in house in IHC so cannot guarantee it. We would recommend antibody Anti-CXCR4 antibody [EPUMBR3] (ab181020) for use in mouse IHC
  • Tested applications
    Suitable for: Flow Cyt, WB, IHC-P, ICC/IF, IHC-Frmore details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mouse, Rat
  • Immunogen

    Synthetic peptide within Human CXCR4 aa 300 to the C-terminus (C terminal). The exact sequence is proprietary.
    (Peptide available as ab155072)

  • Positive control
    • WB: HeLa, Jurkat and WI-38 cell lysates; HEK239 transfected with CXCR4, cell lysate. IF/ICC: Jurkat cells. IHC-P: Human cervical carcinoma, bladder cancer tissue, ovarian adenocarcinoma tissue and tonsil tissue. FC: Jurkat cells
  • General notes

    Our internal data indicates that mouse and rat are not recommended for IHC.

    Abcam recommended secondaries - Goat Anti-Rabbit HRP (ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (ab150077).

    See other anti-rabbit secondary antibodies that can be used with this antibody.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Applications

Our Abpromise guarantee covers the use of ab124824 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.
WB 1/100. Predicted molecular weight: 39 kDa.Can be blocked with CXCR4 peptide (ab155072).

Being CXCR4 a membrane protein, we recommend to not boil the samples after the lysis (before loading the samples on the WB gel).

IHC-P 1/500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

ICC/IF Use a concentration of 5 µg/ml.
IHC-Fr Use at an assay dependent concentration. PubMed: 28932900

Target

  • Function
    Receptor for the C-X-C chemokine CXCL12/SDF-1 that transduces a signal by increasing intracellular calcium ions levels and enhancing MAPK1/MAPK3 activation. Acts as a receptor for extracellular ubiquitin; leading to enhance intracellular calcium ions and reduce cellular cAMP levels. Involved in haematopoiesis and in cardiac ventricular septum formation. Plays also an essential role in vascularization of the gastrointestinal tract, probably by regulating vascular branching and/or remodeling processes in endothelial cells. Could be involved in cerebellar development. In the CNS, could mediate hippocampal-neuron survival. Acts as a coreceptor (CD4 being the primary receptor) for HIV-1 X4 isolates and as a primary receptor for some HIV-2 isolates. Promotes Env-mediated fusion of the virus.
  • Tissue specificity
    Expressed in numerous tissues, such as peripheral blood leukocytes, spleen, thymus, spinal cord, heart, placenta, lung, liver, skeletal muscle, kidney, pancreas, cerebellum, cerebral cortex and medulla (in microglia as well as in astrocytes), brain microvascular, coronary artery and umbilical cord endothelial cells. Isoform 1 is predominant in all tissues tested.
  • Involvement in disease
    Defects in CXCR4 are a cause of WHIM syndrome (WHIM) [MIM:193670]; also known as warts, hypogammaglobulinemia, infections and myelokathexis. WHIM syndrome is an immunodeficiency disease characterized by neutropenia, hypogammaglobulinemia and extensive human papillomavirus (HPV) infection. Despite the peripheral neutropenia, bone marrow aspirates from affected individuals contain abundant mature myeloid cells, a condition termed myelokathexis.
  • Sequence similarities
    Belongs to the G-protein coupled receptor 1 family.
  • Domain
    The amino-terminus is critical for ligand binding. Residues in all four extracellular regions contribute to HIV-1 coreceptor activity.
  • Post-translational
    modifications
    Phosphorylated on agonist stimulation. Rapidly phosphorylated on serine and threonine residues in the C-terminal. Phosphorylation at Ser-324 and Ser-325 leads to recruitment of ITCH, ubiquitination and protein degradation.
    Ubiquitinated by ITCH at the cell membrane on agonist stimulation. The ubiquitin-dependent mechanism, endosomal sorting complex required for transport (ESCRT), then targets CXCR4 for lysosomal degradation. This process is dependent also on prior Ser-/Thr-phosphorylation in the C-terminal of CXCR4. Also binding of ARRB1 to STAM negatively regulates CXCR4 sorting to lysosomes though modulating ubiquitination of SFR5S.
    Sulfation on Tyr-21 is required for efficient binding of CXCL12/SDF-1alpha and promotes its dimerization.
    O- and N-glycosylated. Asn-11 is the principal site of N-glycosylation. There appears to be very little or no glycosylation on Asn-176. N-glycosylation masks coreceptor function in both X4 and R5 laboratory-adapted and primary HIV-1 strains through inhibiting interaction with their Env glycoproteins. The O-glycosylation chondroitin sulfate attachment does not affect interaction with CXCL12/SDF-1alpha nor its coreceptor activity.
  • Cellular localization
    Cell membrane. In unstimulated cells, diffuse pattern on plasma membrane. On agonist stimulation, colocalizes with ITCH at the plasma membrane where it becomes ubiquitinated.
  • Information by UniProt
  • Database links
  • Alternative names
    • C-X-C chemokine receptor type 4 antibody
    • CD184 antibody
    • CD184 antigen antibody
    • Chemokine (C X C motif) receptor 4 antibody
    • Chemokine CXC Motif Receptor 4 antibody
    • CXC-R4 antibody
    • CXCR-4 antibody
    • CXCR4 antibody
    • CXCR4_HUMAN antibody
    • D2S201E antibody
    • FB22 antibody
    • Fusin antibody
    • HM89 antibody
    • HSY3RR antibody
    • LAP 3 antibody
    • LAP3 antibody
    • LCR1 antibody
    • LESTR antibody
    • Leukocyte derived seven transmembrane domain receptor antibody
    • Leukocyte-derived seven transmembrane domain receptor antibody
    • Lipopolysaccharide associated protein 3 antibody
    • Neuropeptide Y receptor Y3 antibody
    • NPY3R antibody
    • NPYR antibody
    • NPYRL antibody
    • NPYY3 antibody
    • NPYY3R antibody
    • Probable G protein coupled receptor lcr1 homolog antibody
    • SDF 1 receptor antibody
    • SDF-1 receptor antibody
    • SEVEN-TRANSMEMBRANE-SEGMENT RECEPTOR antibody
    • Stromal cell derived factor 1 receptor antibody
    • Stromal cell-derived factor 1 receptor antibody
    • WHIM antibody
    • WHIMS antibody
    see all

Images

  • Immunohistochemical detection of CXCR4 expression in human tissue specimens of normal appearance

    CXCR4 was detected in the indicated PFA-fixed, paraffin-embedded human tissues using ab124824 at 5 μ/ml overnight at 4°C.

    A, kidney. B, adrenal gland. C, cerebellum. D, bone marrow, brown staining: CXCR4, green staining: CD45. E, Spleen. F, testis. G, lung. H, colon.

  • Flow Cytometry analysis of Jurkat (human T cell leukemia T lymphocyte) cells labeling CXCR4 with purified ab124824 at 1:260 dilution (10 µg/ml) - Red. Cells were fixed with 4% paraformaldehyde . A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) - Black. Unlabeled control - Blue. Untreated cells - Green
  • All lanes : Anti-CXCR4 antibody [UMB2] (ab124824)

    Lane 1 : CHO (negative control)
    Lane 2 : Jurkat whole cell
    Lane 3 : Jurkat membrane
    Lane 4 : Jurkat nuclear (negative control)

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat anti-rabbit at 1/10000 dilution

    Predicted band size: 39 kDa
    Observed band size: 41 kDa
    why is the actual band size different from the predicted?



    Running buffer: MOPS.

    Conditions: denatured/reduced.

    This blot was produced using a 4-12% Bis-Tris gel under the MOPS buffer system. The gel was run at 200V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab124824 (anti-CXCR4) and ab7671 (loading ctrl), overnight at 4°C. Before imaging, antibody binding was detected using labelled goat anti-rabbit (H+L; green) and labelled goat anti-mouse (H+L; red) at  1:10,000 dilutions for 1hr at room temperature.

  • Characterization of UMB-2 (ab124824) by immunofluorescent staining of transfected cells. HEK-293 cells expressing CCR7 or CXCR4 were either not exposed or exposed to 100 ng/ml MIP-3 or 100 ng/ml SDF-1 for 30 min, subsequently fixed and immunofluorescently stained with 1 µg/ml anti-CCR7 {1188} or anti-CXCR4 {UMB-2} at a dilution of 1∶100. Note that UMB-2 detected prominent immunofluorescence at the level of the plasma membrane only in CXCR4- but not in CCR7-expressing cells, and that SDF-1 exposure induced a rapid translocation of CXCR4 receptor immunostaining from the plasma membrane into the cytosol. Representative results from one of three independent experiments are shown. Scale bar, 20 µm.

  • Unpurified ab124824, at 1/50 dilution, staining CXCR4 in paraffin-embedded Human cervical carcinoma tissue by immunohistochemistry.

  • Immunofluorescence staining of Jurkat cells with purified ab124824 at a working dilution of 1 in 250, counter-stained with DAPI. Tubulin was stained with mouse anti-tubulin at a dilution of 1/1000 (ab7291) and Alexa Fluor® 594 goat anti-mouse at a dilution of 1/500 (ab150120) . The secondary antibody was ab150077 Alexa Fluor® 488 goat anti rabbit, used at a dilution of 1 in 500. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in the bottom middle and right hand panels - for the first negative control, purified ab124824 was used at a dilution of 1/200 followed by an Alexa Fluor® 555 goat anti-mouse antibody at a dilution of 1/500 and for the second negative control mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab15007) were used.

  • Western blot analysis of the specificity of anti-CXCR4 antibodies. Membrane preparations from HEK-293 cells stably transfected to express either CCR7 or CXCR4 were separated on 10% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes. Membranes were then incubated with affinity-purified 1 µg/ml anti-CCR7 {1188} or anti-CXCR4 {UMB-2} hybridoma supernatant at a dilution of 1:100. Blots were developed using enhanced chemiluminescence. Note that UMB-2 detected a band of the expected molecular weight only in CXCR4- but not in CCR7-transfected cells. Two additional experiments gave similar results.

  • All lanes : Anti-CXCR4 antibody [UMB2] (ab124824) at 1/1000 dilution

    Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
    Lanes 2-3 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 39 kDa
    Observed band size: 43 kDa why is the actual band size different from the predicted?


    Exposure time: 1 second


    Blocking and diluting buffer: 5% NFDM/TBST

    We suggest to not boil the sample after lysis.

  • Anti-CXCR4 antibody [UMB2] (ab124824) at 1/100 dilution (purified) + WI-38 cell lysate at 10 µg

    Secondary
    HRP goat anti-rabbit (H+L) at 1/1000 dilution

    Predicted band size: 39 kDa
    Observed band size: 43 kDa why is the actual band size different from the predicted?



    Blocking buffer: 5% NFDM/TBST

    Dilution buffer: 5% NFDM/TBST

  • Immunohistochemical staining of paraffin embedded human bladder cancer with purified ab124824 at a working dilution of 1/500. The secondary antibody used is ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L), at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

  • ab124824 stained Jurkat cells. The cells were 100% methanol fixed for 5 minutes at -20°C and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab124824 at 5ug/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

  • Unpurified ab124824, at 1/50 dilution, staining CXCR4 in paraffin-embedded Human ovarian adenocarcinoma tissue by immunohistochemistry.

  • Unpurified ab124824, at 1/50 dilution, staining CXCR4 in paraffin-embedded Human tonsil tissue by immunohistochemistry.

  • Lane 1 : Anti-CXCR4 antibody [UMB2] (ab124824) at 1/500 dilution (unpurified)
    Lane 2 : Anti-CXCR4 antibody [UMB2] (ab124824) at 1/500 dilution

    Lane 1 : HEK239 transfected with a CXCR4 (mouse) expression vector cell lysate
    Lane 2 : HEK239 transfected with an empty expression vector cell lysate

    Lysates/proteins at 100000 cells per lane.

    Secondary
    All lanes : HRP-conjugated Goat anti-rabbit IgG polyclonal at 1/50000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 39 kDa
    Observed band size: 42,47 kDa why is the actual band size different from the predicted?


    Exposure time: 10 seconds

    See Abreview

References

This product has been referenced in:
  • Habringer S  et al. Dual Targeting of Acute Leukemia and Supporting Niche by CXCR4-Directed Theranostics. Theranostics 8:369-383 (2018). Read more (PubMed: 29290814) »
  • Wang R  et al. Quercetin Inhibits Breast Cancer Stem Cells via Downregulation of Aldehyde Dehydrogenase 1A1 (ALDH1A1), Chemokine Receptor Type 4 (CXCR4), Mucin 1 (MUC1), and Epithelial Cell Adhesion Molecule (EpCAM). Med Sci Monit 24:412-420 (2018). Read more (PubMed: 29353288) »
See all 80 Publications for this product

Customer reviews and Q&As

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1-10 of 14 Abreviews

Application
Western blot
Sample
Human Cell lysate - whole cell (Kidney cells (clear cell renal carcinoma))
Gel Running Conditions
Reduced Denaturing (10)
Loading amount
50 µg
Specification
Kidney cells (clear cell renal carcinoma)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%

Paulo Rodrigues

Verified customer

Submitted Aug 10 2018

Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (hepatocytes)
Permeabilization
Yes - 0.1% Triton X-100 in TBS for 10 minutes
Specification
hepatocytes
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 23°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Feb 03 2017

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Monkey Cell (Kidney)
Permeabilization
Yes - 0.1% triton x-100
Specification
Kidney
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jun 10 2016

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Brain)
Permeabilization
No
Specification
Brain
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Mar 04 2016

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Heart)
Permeabilization
Yes - 0.2% triton TX-100
Specification
Heart
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Feb 24 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (BEL-7404)
Gel Running Conditions
Non-reduced Denaturing (10%)
Loading amount
40 µg
Treatment
80µM Doxorubicin for 24hrs
Specification
BEL-7404
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Dr. guoqing zhu

Verified customer

Submitted Jan 20 2016

Application
Western blot
Sample
Mouse Tissue lysate - whole (Heart)
Gel Running Conditions
Reduced Denaturing (10% Invitrogen gel)
Loading amount
40 µg
Specification
Heart
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Mr. Dan Bromage

Verified customer

Submitted Dec 30 2015

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (1BR3.G fibrblast)
Gel Running Conditions
Reduced Denaturing (10%)
Loading amount
50 µg
Specification
1BR3.G fibrblast
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5%

Abcam user community

Verified customer

Submitted Jul 22 2015

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (1BR3.G fibroblast)
Permeabilization
Yes - Triton 1%
Specification
1BR3.G fibroblast
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: rt°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Jul 22 2015

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (colon)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: EDTA, pH8, 100C, 20 min
Permeabilization
No
Specification
colon
Blocking step
3% H2O2 as blocking agent for 10 minute(s) · Concentration: 3% · Temperature: 25°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Oct 27 2014

1-10 of 14 Abreviews

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