Anti-CXCR4 Antibody

Western Blot Validation of CXCR4 in HeLa Cells
Loading: 15 μg of lysates per lane. Antibodies: A00031-1 (1 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.

Independent Antibody Validation (IAV) via Protein Expression Profile in Cell Lines
Loading: 15 μg of lysates per lane. Antibodies: A00031-1 (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.

Validation with CXCR4 siRNA Knockdown in HeLa Cells
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: A00031-1 (2 μg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.

Animal Species Reactivity
Loading: Lysates/proteins at 20 μg per lane. Antibodies: A00031-1 (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.

Recombinant Protein Test
Loading: CXCR4 partial recombinant protein (Novus Biologicals, Cat# H00007852-Q01). Lane 1: Anti-CXCR4 antibody (0.1 μg/mL) 1 h incubation at RT in 5% NFDM/TBST. Lane 2: Coomassie blue staining. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution.

Immunofluorescence Validation of CXCR4 in HeLa Cells
Immunofluorescent analysis of 4% paraformaldehyde-fixed HeLa cells labeling CXCR4 with A00031-1 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red). Image showing both membrane and cytoplasmic staining on HeLa cells.

Flow Cytometry Validation of CXCR4 in HeLa Cells
Overlay histogram showing HeLa cells stained with A00031-1 (red line, 1μg/1x106 cells). 1 h incubation at 4˚C in 2% FBS/PBS. Followed by secondary antibody 488 goat anti-rabbit IgG (H+L) at 1/500 dilution for 1 h 4˚C.

Isotype control antibody (Green line) was mouse IgG1 (1μg/1x106 cells) used under the same conditions.

Overexpression Validation of CXCR4 (Kozak et al., 2002)
U87MG and U87MG-CXCR4 extracts were included as negative and positive controls, respectively, for CXCR4 detection with anti-CXCR4 antibodies.

WB Validation of CXCR4 in Human Metastatic Melanoma (Scala et al., 2006)
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.

Immunohistochemistry Validation of CXCR4 in Human Spleen
Immunohistochemical analysis of paraffin-embedded human spleen tissue using anti-CXCR4 antibody (A00031-1) 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.

Immunocytochemistry Validation of CXCR4 in HeLa Cells
Immunocytochemical analysis of HeLa cells using anti-CXCR4 antibody (A00031-1) at 2 μg/ml. Cells 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.

KO Validation of CXCR4 by Flow Cytometry (?demis, et al., 2010)
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).