Anti-CXCR4 antibody [UMB2]

• IHC-P

Unknown

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 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.

• ICC/IF

PubMed

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.

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/

• IHC-P

Unknown

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.

• IHC-P

Unknown

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.

• IHC-P

Unknown

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.

• ICC/IF

Unknown

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 (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.

• IHC-P

PubMed

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.

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/

• Flow Cyt (Intra)

Lab

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, ab150081) at 1/3000 dilution was used as the secondary antibody. No cross-reactivity with CXCR1/CXCR2/CXCR3/CXCR5.

• ICC/IF

Lab

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 (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.

• Flow Cyt (Intra)

Unknown

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

• IHC-P

PubMed

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.

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/

• Flow Cyt (Intra)

Lab

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCR4 antibody [UMB2] (AB124824)

Immunohistochemical analysis of Paraffin-embedded sections Mouse lung tissue labelling CXCR4 with ab124824 at 1/1000 dilution, followed by LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic and membranous staining on mouse lung tissue is observed. Counter stained with Hematoxylin. Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is LeicaDS9800 (Bond™ Polymer Refine Detection).

The section was incubated with ab124824 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CXCR4 antibody [UMB2] (AB124824)

Immunohistochemical analysis of Paraffin-embedded sections Mouse spleen tissue labelling CXCR4 with ab124824 at 1/1000 dilution, followed by LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic and membranous staining on mouse spleen tissue is observed. Counter stained with Hematoxylin. Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is LeicaDS9800 (Bond™ Polymer Refine Detection).

The section was incubated with ab124824 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• WB

Unknown

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

false

• WB

PubMed

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

false

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/.

• WB

Lab

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 39 kDa

Observed band size: 43 kDa

false

Exposure time: 1s

• WB

AbReview39488****

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

Predicted band size: 39 kDa

Observed band size: 42 kDa,47 kDa

true

Exposure time: 10s

This image is courtesy of an anonymous Abreview

• WB

Unknown

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 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

false