Clone:
REA733
Type of antibody:
Primary antibodies, Recombinant antibodies
Isotype:
recombinant human IgG1
Applications:
FC, MICS, IF, IHC, MC
Alternative names:
Bp50, CD40L receptor, CDW40

Extended validation for CD40 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 REA733
5C3++
HB14+
Cells were incubated with an excess of purified unconjugated CD40 (REA733) 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 CD40 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD40-PE (REA733, red) and counterstained with DRAQ5 (blue) as DNA stain.
View details
Fluorescence microscopy image of CD40 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD40-PE (REA733, red) and counterstained with DRAQ5 (blue) as DNA stain.
Fluorescence microscopy image of CD40 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD40-PE (REA733, red) and counterstained with DRAQ5 (blue) as DNA stain.
View details
Overlay histogram showing flow cytometric analysis of CD40 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD40-PE, clone (REA733). 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 CD40 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD40-PE, clone (REA733). 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 CD40. Human peripheral blood mononuclear cells (PBMCs) were stained with CD40 antibodies and with a suitable counterstaining. As a control, CD40 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 CD40. Human peripheral blood mononuclear cells (PBMCs) were stained with CD40 antibodies and with a suitable counterstaining. As a control, CD40 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 CD40. Human peripheral blood mononuclear cells (PBMCs) were stained with CD40 antibodies and with a suitable counterstaining. As a control, CD40 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 CD40. Human peripheral blood mononuclear cells (PBMCs) were stained with CD40 antibodies and with a suitable counterstaining. As a control, CD40 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 CD40 (REA733). 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 CD40 (REA733). 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 CD40 (REA733). 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 CD40 Antibody, anti-human, REAfinity™

Overview

Clone REA733 recognizes the human CD40 antigen, a transmembrane receptor of the tumor necrosis factor gene superfamily. CD40 is a costimulatory protein constitutively expressed by antigen presenting cells, including dendritic cells, B cells, and macrophages and on endothelial, smooth muscle cells, and fibroblasts. The binding of CD40 to CD154 (CD40L) on T helper cells activates antigen presenting cells and enhances the expression of cytokines, chemokines, matrix metalloproteinases, growth factors, and adhesion molecules. CD40 has been shown to interact with members of the TRAF family.
Additional information: Clone REA733 displays negligible binding to Fc receptors.

Alternative names

Bp50, CD40L receptor, CDW40

Detailed product information

Technical specifications

CloneREA733
Clonalitymonoclonal
Isotyperecombinant human IgG1
Isotype controlREA Control Antibody (S), human IgG1
Hostcell line
Type of antibodyPrimary antibodies, Recombinant antibodies
Specieshuman, non-human primate
Cross-reactivity
cynomolgus monkey (
Macaca fascicularis
)
,
rhesus monkey (
Macaca mulatta
)
, baboon,
chimpanzee (
Pan troglodytes
)
,
squirrel monkey (
Saimiri sciureus
)
AntigenCD40
Alternative names of antigenBp50, CD40L receptor, CDW40
Molecular mass of antigen [kDa]28
Distribution of antigenB cells, macrophages, fibroblasts, smooth muscle
Entrez Gene ID958
RRIDAB_2657997, AB_2657998, AB_2657999, AB_2658000, AB_2658001, AB_2658002, AB_2658003, AB_2658004, AB_2658005, AB_2658006, AB_2658007, AB_2811385, AB_2657996

Resources for CD40 Antibody, anti-human, REAfinity™

Certificates

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

References for CD40 Antibody, anti-human, REAfinity™

Publications

  1. Stamenkovic, I. et al. (1989) A B-lymphocyte activation molecule related to the nerve growth factor receptor and induced by cytokines in carcinomas. EMBO J. 8(5): 1403-1410
  2. Sato, T. et al. (1995) A novel member of the TRAF family of putative signal transducing proteins binds to the cytosolic domain of CD40. FEBS Lett. 358(2): 113-118
  3. Chatzigeorgiou, A. et al. (2009) CD40/CD40L signaling and its implication in health and disease. Biofactors 35(6): 474-483

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