CXCR4 Antibody

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ProSci
Product Code: PSI-1012
Product Group: Primary Antibodies
Supplier: ProSci
CodeSizePrice
PSI-1012-0.02mg0.02mg£150.00
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PSI-1012-0.1mg0.1mg£449.00
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Overview

Host Type: Rabbit
Antibody Isotype: IgG
Antibody Clonality: Polyclonal
Regulatory Status: RUO
Target Species:
  • Human
  • Mouse
  • Rat
Applications:
  • Enzyme-Linked Immunosorbent Assay (ELISA)
  • Immunofluorescence (IF)
  • Western Blot (WB)

Images

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<strong>Figure 1 Western Blot Validation of CXCR4 in HeLa Cells </strong><br> Loading: 15 μg of lysates per lane. Antibodies: 1012 (1 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
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<strong>Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile</strong><br> Loading: 15 μg of lysates per lane. Antibodies: 1009 (1 μg/mL), 1012 (1 μg/mL), and beta-actin (1 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
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<strong>Figure 3 Validation with CXCR4 siRNA Knockdown in HeLa Cells </strong><br> HeLa cells were transfected with control siRNAs (lane 1) or CXCR4 siRNAs (lane 2) Loading: 10 μg of HeLa whole cell lysates per lane. Antibodies: 1012 (2 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
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<strong>Figure 4 Animal Species Reactivity </strong><br> Loading: Lysates/proteins at 20 μg per lane. Antibodies: 1009 (2 μg/mL) or 1012 (2 μg/mL). 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
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<strong>Figure 5 Immunofluorescence Validation of CXCR4 </strong><br>Immunofluorescent analysis of 4% paraformaldehyde-fixed HeLa cells labeling CXCR4 with 1012 at 4 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red). Image showing both membrane and cytoplasmic staining on HeLa cell line.
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<strong>Figure 6 Immunohistochemistry Validation of CXCR4 in Human Spleen</strong><br> Immunohistochemical analysis of paraffin-embedded human spleen tissue using anti-CXCR4 antibody (1012) at 5 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚ C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
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<strong>Figure 7 KO Validation of CXCR4 by Flow Cytometry (?demis, et al., 2010) </strong><br> Astrocytes from wild-type or CXCR4 knockout mice were stained with primary antibodies against CXCR4 and FITC-labeled secondary antibodies, and subsequently subjected to flow cytometry. CXCR4-/- astrocytes (red) showed loss of CXCR4 cell-surface expression compared with wild-type cells (black).
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<strong>Figure 8 Overexpression Validation of CXCR4 (Kozak et al., 2002) </strong><br> U87MG and U87MG-CXCR4 extracts were included as negative and positive controls, respectively, for CXCR4 detection with anti-CXCR4 antibodies.
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<strong>Figure 9 WB Validation of CXCR4 in Human Metastatic Melanoma (Scala et al., 2006) </strong><br> CXCR4 protein was detected in the human metastatic melanoma cell lines and human melanoma cell line (colo38), but not in the human primary melanocytes (MPR1) with anti-CXCR4 antibodies.
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<strong>Figure 1 Western Blot Validation of CXCR4 in HeLa Cells </strong><br> Loading: 15 μg of lysates per lane. Antibodies: 1012 (1 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile</strong><br> Loading: 15 μg of lysates per lane. Antibodies: 1009 (1 μg/mL), 1012 (1 μg/mL), and beta-actin (1 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 3 Validation with CXCR4 siRNA Knockdown in HeLa Cells </strong><br> HeLa cells were transfected with control siRNAs (lane 1) or CXCR4 siRNAs (lane 2) Loading: 10 μg of HeLa whole cell lysates per lane. Antibodies: 1012 (2 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 4 Animal Species Reactivity </strong><br> Loading: Lysates/proteins at 20 μg per lane. Antibodies: 1009 (2 μg/mL) or 1012 (2 μg/mL). 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.
<strong>Figure 5 Immunofluorescence Validation of CXCR4 </strong><br>Immunofluorescent analysis of 4% paraformaldehyde-fixed HeLa cells labeling CXCR4 with 1012 at 4 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red). Image showing both membrane and cytoplasmic staining on HeLa cell line.
<strong>Figure 6 Immunohistochemistry Validation of CXCR4 in Human Spleen</strong><br> Immunohistochemical analysis of paraffin-embedded human spleen tissue using anti-CXCR4 antibody (1012) at 5 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚ C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
<strong>Figure 7 KO Validation of CXCR4 by Flow Cytometry (?demis, et al., 2010) </strong><br> Astrocytes from wild-type or CXCR4 knockout mice were stained with primary antibodies against CXCR4 and FITC-labeled secondary antibodies, and subsequently subjected to flow cytometry. CXCR4-/- astrocytes (red) showed loss of CXCR4 cell-surface expression compared with wild-type cells (black).
<strong>Figure 8 Overexpression Validation of CXCR4 (Kozak et al., 2002) </strong><br> U87MG and U87MG-CXCR4 extracts were included as negative and positive controls, respectively, for CXCR4 detection with anti-CXCR4 antibodies.
<strong>Figure 9 WB Validation of CXCR4 in Human Metastatic Melanoma (Scala et al., 2006) </strong><br> CXCR4 protein was detected in the human metastatic melanoma cell lines and human melanoma cell line (colo38), but not in the human primary melanocytes (MPR1) with anti-CXCR4 antibodies.

Documents

Further Information

Additional Names:
CXCR4 Antibody: FB22, HM89, LAP3, LCR1, NPYR, WHIM, CD184, LESTR, NPY3R, NPYRL, HSY3RR, NPYY3R, D2S201E
Application Note:
WB: 1 - 2 μg/mL; IHC-P: 5 μg/mL; IF: 4 μg/mL.

Antibody validated: Western Blot in human, mouse, and rat samples; Immunohistochemistry and Immunofluorescence in human samples. All other applications and species not yet tested.
Background:

CXCR4, a G-protein coupled receptor (GPCR) with seven transmembrane domains, is a CXC chemokine receptor specific for stromal-derived-factor-1 (SDF-1 or CXCL12). CXCR4 was initially discovered as one of the co-receptors for HIV entry into CD4+ T cells (1). Blocking CXCR4 could be potentially used as novel therapeutics for HIV treatment.

CXCR4 signaling plays an important role in the migration, proliferation and quiescence of hematopoietic stem cell and their retention within the bone marrow, where it has high levels of SDF-1/CXCL12(2). It has been demonstrated that CXCR4 signaling mediates CD20 up-regulation on B cells (3).

CXCR4 is highly expressed in more than 23 types of cancer, including breast cancer, ovarian cancer, melanoma, and prostate cancer, while there is very less or no expression of CXCR4 in healthy tissues. CXCR4 expression in cancer cells has been reported to be associated with tumor survival, growth and metastasis in tissues with high levels of SDF-1/CXCL12, such as lungs, liver and bone marrow (4,5).

CXCR4 has been shown to regulate neuronal migration, cell positioning and axon wiring (6,7). CXCR4 mutant mice displayed aberrant neuronal distribution, which implicates the role in neuronal disorders such as epilepsy. CXCR4 is also involved in WHIM syndrome (8). WHIM mutations in CXCR4 were recently found in patients with Waldenstrom's macroglobulinemia, and these mutations are correlated to clinical resistance to ibrutinib (9,10).

Background References:
  • Dimitrov DS. Cell 1997;91:721-730
  • Chen et al. Circ Res. 2010;107:1083-93
  • Pavlasova et al. Blood 2016; 128: 1609-13
  • Sun et al. Cancer Metastasis Reviews 2010;29:709-22
  • Balkwill F. Nature Reviews 2004;4:540-50
  • Bagri et al. Development 2002; 129:4249-60
  • Yang et al. Development 2013;140:4554-64
  • Balabanjian et al. The Journal of Clinical Investigation 2008; 118:1074-84
  • Hunter et al. Blood 2014;123:1637-46
  • Treon et al. N. Engl. J. Med. 2015;372:1430-40
Buffer:
CXCR4 Antibody is supplied in PBS containing 0.02% sodium azide.
Concentration:
1 mg/mL
Conjugate:
Unconjugated
DISCLAIMER:
Optimal dilutions/concentrations should be determined by the end user. The information provided is a guideline for product use. This product is for research use only.
Homology:
Predicted species reactivity based on immunogen sequence: Pig: (100%), Bovine: (86%), Sheep: (86%)
Immunogen:
Anti-CXCR4 antibody (1012) was raised against a peptide corresponding to 15 amino acids near the center of human CXCR4 isoform b.

The immunogen is located within amino acids 170-220 of CXCR4.
ISOFORMS:
Human CXCR4 has four isoforms, including isoform a (356aa, 40.2kD), isoform b (352aa, 39.7kD), isoform c (423aa, 47.6kD), and isoform d (385aa, 43.4kD). This antibody detects human isoform b only, but not other human isoforms. Mouse CXCR4 has two isoforms, including isoform 1 (359aa, 40.4kD) and isoform 2 (357aa, 40.2kD). Rat CXCR4 has only one isoform identified so far (349aa, 39.4kD).
NCBI Gene ID #:
7852
NCBI Official Name:
chemokine (C-X-C motif) receptor 4
NCBI Official Symbol:
CXCR4
NCBI Organism:
Homo sapiens
Physical State:
Liquid
PREDICTED MOLECULAR WEIGHT:
Predicted: 40kD

Observed: 44kD (Post-modification: 2 N-linked glycosylation)
Protein Accession #:
NP_003458
Protein GI Number:
4503175
Purification:
CXCR4 Antibody is Protein A purified.
Research Area:
Chemokines & Cytokines,Stem Cell,Infectious Disease
Swissprot #:
P61073
User NOte:
Optimal dilutions for each application to be determined by the researcher.
VALIDATION:

Independent Antibody Validation (Figure 2) shows similar CXCR4 expression profile in both human and mouse cell lines detected by two independent anti-CXCR4 antibodies that recognize different epitopes, 1009 against N-terminus ectodomain and 1012 against the second extracellular loop.  CXCR4 proteins are detected in most of the cell lines, but not in THP-1 cells by the two independent antibodies.  Additionally, Figure 2 shows the mouse CXCR4 protein in NIH/3T3 and C2C12 cell lines migrates slightly slower than human isoform b detected by both CXCR4 antibodies (1009 and 1012), which is well correlated with their calculated molecular masses (40.4 kDa vs 39.7 kDa).  

siRNA knockdown validation (Figure 3): Anti-CXCR4 antibody (1012) specificity was further verified by CXCR4 specific siRNA knockdown. CXCR4 signal in HeLa cells transfected with CXCR4 siRNAs was weaker in comparison with that in HeLa cells transfected with control siRNAs.

Animal Species Reactivity (Figure 4): Anti-CXCR4 antibodies (1009 and 1012) can detect the expression of CXCR4 protein in rat thymus, but not in rat brain and rat heart.

References

  1. Odemis et al. CXCR7 is an active component of SDF-1 signalling in astrocytes and Schwann cells. J Cell Sci. 2010;123(Pt 7):1081-8. PMID: 20197403
  2. Kozak et al. Segregation of CD4 and CXCR4 into distinct lipid microdomains in T lymphocytes suggests a mechanism for membrane destabilization by human immunodeficiency virus. J Virol. 2002;76(4):1802-15.PMID: 11799176
  3. Scala et al. Human melanoma metastases express functional CXCR4. Clin Cancer Res 2006;12(8):2427-33. PMID: 16638848
  4. Recasens-Zorzo et al. Pharmacological modulation of CXCR4 cooperates with BET bromodomain inhibition in diffuse large B-cell lymphoma.Haematologica. PMID: 29954928
  5. Pandit et al. Chronic allergen challenge induces pulmonary extramedullary hematopoiesis. Exp Lung Res. 2011;37(5):279-90. PMID: 21309736
  6. Nobuko et al. microRNA-150 regulates mobilization and migration of bone marrow-derived mononuclear cells by targeting CXCR4. PLoS ONE 2011; 6(10):e23114. PMID: 22039399
  7. Nimmagadda et al. Immunoimaging of CXCR4 expression in brain tumor xenografts using SPECT/CT. J Nucl Med. 2009;50(7):1124-30.PMID: 19525448
  8. Lee et al. Gene expression in temporal lobe epilepsy is consistent with increased release of glutamate by astrocytes. Mol Med. 2007;13(1-2):1-13. PMID: 17515952
  9. Terlika et al. Sustained exposure to nicotine leads to extramedullary hematopoiesis in the spleen. Stem Cells 2006;24:2373-81. PMID: 16825610
  10. Schabath et al. CD24 affects CXCR4 function in pre-B lymphocytes and breast carcinoma cells. J Cell Sci. 2006;119(Pt 2):314-25. PMID: 16390867

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