Recombinant 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 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, ab150081) at 1/3000 dilution was used as the secondary antibody.

No cross-reactivity with CXCR1/CXCR2/CXCR3/CXCR5.

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, ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) - Black. Unlabeled control - Blue. Untreated cells - Green

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. ab150077 lexaFluor^®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.

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.

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.

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. ab150077 lexaFluor^®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.

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.

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.

Blocking and diluting buffer: 5% NFDM/TBST

We suggest to not boil the sample after lysis.

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.

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

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.

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