Clone:
REA923
Type of antibody:
Primary antibodies, Recombinant antibodies
Isotype:
recombinant human IgG1
Applications:
FC
Alternative names:
Ephrin type-B receptor 4, Hepatoma transmembrane kinase, Tyrosine-protein kinase TYRO11, HTK, MYK1, TYRO11

Extended validation for EPHB4 Antibody, anti-human, REAfinity™

Specificity

Epitope competition
In order to compare the epitope specificity of an antibody, the clone being used is compared with other known clones recognizing the same antigen in a competition assay.
Other clonesOverlap in epitope recognition with REA923
001+
04-
Cells were incubated with an excess of purified unconjugated EPHB4 (REA923) antibody followed by staining with fluorochrome-conjugated antibodies of other known clones against the same marker. Based on the fluorescence signal obtained, the clones were identified as recognizing completely overlapping (++), partially overlapping (+), or completely different epitopes (-) of the marker.
Knockout validation
To ensure antibody specificity, the target gene is knocked out in a suitable cell line using the CRISPR/Cas9 system and the knockout is confirmed by sequencing of the target locus. The antibody is considered to bind specifically to the intended epitope if no antibody binding to the knockout cells can be detected. The antibody staining is controlled by fluorescence microscopy and/or flow cytometry.
WT
KO
View details
Fluorescence microscopy image of EPHB4 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with EPHB4-PE (REA923, red) and counterstained with DRAQ5 (blue) as DNA stain.
View details
Fluorescence microscopy image of EPHB4 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with EPHB4-PE (REA923, red) and counterstained with DRAQ5 (blue) as DNA stain.
Fluorescence microscopy image of EPHB4 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with EPHB4-PE (REA923, red) and counterstained with DRAQ5 (blue) as DNA stain.
View details
Overlay histogram showing flow cytometric analysis of EPHB4 knockout cells. Wild type (red) and knockout cells (blue) were stained with EPHB4-PE, clone (REA923). Flow cytometry was performed with the MACSQuant
®
Analyzer. Cell debris, dead cells and cell doublets were excluded from the analysis based on scatter signals and propidium iodide fluorescence.
Overlay histogram showing flow cytometric analysis of EPHB4 knockout cells. Wild type (red) and knockout cells (blue) were stained with EPHB4-PE, clone (REA923). Flow cytometry was performed with the MACSQuant
®
Analyzer. Cell debris, dead cells and cell doublets were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

Sensitivity

Performance comparison
Selected fluorochrome conjugated antibodies from Miltenyi Biotec were compared to commercially available hybridoma clones in flow cytometry analysis.
View details
Flow cytometric comparison of different clones for EPHB4. A mixture of negative Human peripheral blood mononuclear cells (PBMCs) and MCF-7 positive cells were stained with EPHB4 antibodies and plotted against the side scatter. As a control, EPHB4 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
View details
Flow cytometric comparison of different clones for EPHB4. A mixture of negative Human peripheral blood mononuclear cells (PBMCs) and MCF-7 positive cells were stained with EPHB4 antibodies and plotted against the side scatter. As a control, EPHB4 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
View details
Flow cytometric comparison of different clones for EPHB4. A mixture of negative Human peripheral blood mononuclear cells (PBMCs) and MCF-7 positive cells were stained with EPHB4 antibodies and plotted against the side scatter. As a control, EPHB4 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
View details
Flow cytometric comparison of different clones for EPHB4. A mixture of negative Human peripheral blood mononuclear cells (PBMCs) and MCF-7 positive cells were stained with EPHB4 antibodies and plotted against the side scatter. As a control, EPHB4 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
Flow cytometric comparison of different clones for EPHB4. A mixture of negative Human peripheral blood mononuclear cells (PBMCs) and MCF-7 positive cells were stained with EPHB4 antibodies and plotted against the side scatter. As a control, EPHB4 antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant® Analyzer. Cell debris, dead cells, and cell doublets were excluded from the analysis based on scatter signals and 4',6-diamidino-2-phenylindole (DAPI) fluorescence. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.
Fixation data
To provide an indication on how an antibody performs after fixation of cells, in-house data on staining results before and after fixation with 3.7% formaldehyde using Miltenyi Biotec antibodies are provided. Different experimental settings may lead to different results.
No fixation
Post fixation
View details
Comparison of staining pattern on non-fixed and fixed cells using EPHB4 (REA923). The performance of the antibody after fixation was tested by comparing the staining pattern on fresh (no fixation) versus fixed (post fixation) cells.
View details
Comparison of staining pattern on non-fixed and fixed cells using EPHB4 (REA923). The performance of the antibody after fixation was tested by comparing the staining pattern on fresh (no fixation) versus fixed (post fixation) cells.
Comparison of staining pattern on non-fixed and fixed cells using EPHB4 (REA923). The performance of the antibody after fixation was tested by comparing the staining pattern on fresh (no fixation) versus fixed (post fixation) cells.

Specifications for EPHB4 Antibody, anti-human, REAfinity™

Overview

Clone REA923 recognizes the human ephrin type-B receptor 4 (EPHB4) antigen, which is a member of the Eph receptor tyrosine kinase family. The ligand for EPHB4 is ephrin-B2. EPHB4 is found in different tissues, e.g., in placenta, kidney, liver, lung, pancreas, skeletal muscle, and heart. It is expressed on myeloid hematopoietic cells and endothelial cells. EPHB4 functions as an inhibitor for cell-cell adhesion, chemotaxis, and angiogenesis as well as a promoter for the differentiation of megakaryocytic and erythroid progenitor cells.
Additional information: Clone REA923 displays negligible binding to Fc receptors.

Alternative names

Ephrin type-B receptor 4, Hepatoma transmembrane kinase, Tyrosine-protein kinase TYRO11, HTK, MYK1, TYRO11

Detailed product information

Technical specifications

CloneREA923
Clonalitymonoclonal
Isotyperecombinant human IgG1
Isotype controlREA Control Antibody (S), human IgG1
Hosthuman cell line
Type of antibodyPrimary antibodies, Recombinant antibodies
Specieshuman
AntigenEPHB4
Alternative names of antigenEphrin type-B receptor 4, Hepatoma transmembrane kinase, Tyrosine-protein kinase TYRO11, HTK, MYK1, TYRO11
Molecular mass of antigen [kDa]107
Distribution of antigenendothelial cells, other, breast, colon, kidney, liver, lung, pancreas, placenta, skeletal muscle
Entrez Gene ID2050
RRIDAB_2727059, AB_2727110, AB_2727060, AB_2727111, AB_2727061, AB_2727255, AB_2727213, AB_2727112, AB_2727062, AB_2727113, AB_2727063, AB_2727108, AB_2727058, AB_2928625, AB_2928624, AB_2727109

Resources for EPHB4 Antibody, anti-human, REAfinity™

Certificates

Please follow this
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to search for Certificates of Analysis (CoA) by lot number.

References for EPHB4 Antibody, anti-human, REAfinity™

Publications

  1. Erber, R. et al. (2006) EphB4 controls blood vascular morphogenesis during postnatal angiogenesis. EMBO J. 25(3): 628-641
  2. Füller, T. et al. (2003) Forward EphB4 signaling in endothelial cells controls cellular repulsion and segregation from ephrinB2 positive cells. J. Cell. Sci. 116(12): 2461-2470

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