Anti-CXCR4 antibody [UMB2]

19 product images

• 

  Fischer, T. et al PLoS One. 2008;3(12):e4069. doi: 10.1371/journal.pone.0004069. Epub 2008 Dec 31 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

  Immunocytochemistry/ Immunofluorescence - Anti-CXCR4 antibody [UMB2] (ab124824)

  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.

• 

  Li X et al., European journal of nuclear medicine and molecular imaging. 45,4 (2018): 558-566. Fig 3. doi:10.1007/s00259-017-3831-0. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCR4 antibody [UMB2] (ab124824)

  
  
  
  
  Immunohistochemistry analysis of cryosections from carotid endarterectomy specimens labeling CXCR4 with ab124824 at 1/300 dilution. CXCR4 expression in a representative inflamed carotid plaque lesion. Brightfield micrographs showed brown chemoimmunoreactive CXCR4 and CD68 (macrophage) staining. Co-localized CXCR4 and CD68 expression was observed in these two adjacent sections.
  
  
  
  

• 

  Fischer, T. et al PLoS One. 2008;3(12):e4069. doi: 10.1371/journal.pone.0004069. Epub 2008 Dec 31. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/.

  Western blot - Anti-CXCR4 antibody [UMB2] (ab124824)

  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: Western blot - Anti-CXCR4 antibody [UMB2] (ab124824)

  Predicted band size: 39 kDa

• 

  Flow Cytometry (Intracellular) - Anti-CXCR4 antibody [UMB2] (ab124824)

  Intracellular 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, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) - Black. Unlabeled control - Blue. Untreated cells - Green

• 

  Immunohistochemistry (Frozen sections) - Anti-CXCR4 antibody [UMB2] (ab124824)

  Immunohistochemical (Frozen) analysis of mouse E14.5 cerebrum labeling CXCR4 with ab124824 at 1/250 (10.8 μg/mL). Sections were fixed with 4% PFA and permeabilised with 0.2% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/mL) was used as the secondary antibody. Nuclei were counterstained blue with DAPI. Heat mediated antigen retrieval was performed using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).
  

  Positive staining on mouse embryonic cerebrum.

• 

  Costa, M.J. et al PLoS One. 2018 Mar 19;13(3):e0194688. doi: 10.1371/journal.pone.0194688. eCollection 2018 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCR4 antibody [UMB2] (ab124824)

  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.

• 

  Western blot - Anti-CXCR4 antibody [UMB2] (ab124824)

  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 Anti-alpha 1 Sodium Potassium ATPase antibody [464.6] 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.

  All lanes: Western blot - Anti-CXCR4 antibody [UMB2] (ab124824)

  Lane 1: CHO (negative control) at 20 µg

  Lane 2: Jurkat whole cell at 20 µg

  Lane 3: Jurkat membrane at 20 µg

  Lane 4: Jurkat nuclear (negative control) at 20 µg

  Secondary

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

  Predicted band size: 39 kDa

  Observed band size: 41 kDa

• 

  This image is courtesy of an anonymous customer review

  Western blot - Anti-CXCR4 antibody [UMB2] (ab124824)

  All lanes: Western blot - Anti-CXCR4 antibody [UMB2] (ab124824) at 1/500 dilution

  Lane 1: HEK239 transfected with a CXCR4 (mouse) expression vector cell lysate at 100000 Cells

  Lane 2: HEK239 transfected with an empty expression vector cell lysate at 100000 Cells

  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 kDa, 47 kDa

  Exposure time: 10s

• 

  Immunohistochemistry (Frozen sections) - Anti-CXCR4 antibody [UMB2] (ab124824)

  Immunohistochemical (Frozen) analysis of rat E14.5 cerebrum labeling CXCR4 with ab124824 at 1/250 (10.8 μg/mL). Sections were fixed with 4% PFA and permeabilised with 0.2% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/mL) was used as the secondary antibody. Nuclei were counterstained blue with DAPI. Heat mediated antigen retrieval was performed using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).
  

  Positive staining on rat embryonic cerebrum.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCR4 antibody [UMB2] (ab124824)

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

  Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-CXCR4 antibody [UMB2] (ab124824)

  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 (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) and Alexa Fluor^® 594 goat anti-mouse at a dilution of 1/500 (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) . The secondary antibody was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) 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 (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) and anti-rabbit secondary antibody (ab15007) were used.

• 

  Western blot - Anti-CXCR4 antibody [UMB2] (ab124824)

  Blocking and diluting buffer: 5% NFDM/TBST
  

  We suggest to not boil the sample after lysis.

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

  Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

  Lanes 2 - 3: Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates at 15 µg

  Secondary

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

  Predicted band size: 39 kDa

  Observed band size: 43 kDa

  Exposure time: 1s

• 

  Western blot - Anti-CXCR4 antibody [UMB2] (ab124824)

  Blocking buffer: 5% NFDM/TBST

  Dilution buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-CXCR4 antibody [UMB2] (ab124824) at 1/100 dilution

  All lanes: WI-38 cell lysate at 10 µg

  Secondary

  All lanes: HRP goat anti-rabbit (H+L) at 1/1000 dilution

  Predicted band size: 39 kDa

  Observed band size: 43 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCR4 antibody [UMB2] (ab124824)

  Immunohistochemical staining of paraffin embedded human bladder cancer with purified ab124824 at a working dilution of 1/500. The secondary antibody used is Goat Anti-Rabbit IgG H&L (HRP) 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.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-CXCR4 antibody [UMB2] (ab124824)

  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 (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.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCR4 antibody [UMB2] (ab124824)

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

  Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCR4 antibody [UMB2] (ab124824)

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

  Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• 

  Flow Cytometry (Intracellular) - Anti-CXCR4 antibody [UMB2] (ab124824)

  Flow cytometry overlay histogram showing left Jurkat positive cells and right negative HepG2 stained with ab124824 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab124824) (1x 106 in 100μl at 1.0μg/ml (1/1900)) for 30min at 22°C.
  
  The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C
  
  Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.
  
  Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.
  
  This antibody gave a positive signal in Jurkat Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• 

  Flow Cytometry (Intracellular) - Anti-CXCR4 antibody [UMB2] (ab124824)

  Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293T (human embryonic kidney) cells transfected with a mix of human CXCR1, CXCR2, CXCR3 and CXCR5 expression vector containing a myc-his tag (Left) / HEK-293T transfected with a human CXCR4 expression vector containing a myc-his tag (Right) labelling CXCR4 with ab124824 at 1/2000 dilution (0.1μg)/ Left and Right. A Goat Anti-Rabbit IgG (Alexa Fluor® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 1/3000 dilution was used as the secondary antibody.
  
  No cross-reactivity with CXCR1/CXCR2/CXCR3/CXCR5.