Clone:
REA499
Type of antibody:
Primary antibodies, Recombinant antibodies
Isotype:
recombinant human IgG1
Applications:
FC, MICS, IF, IHC, MC
Alternative names:
S152, TNFRSF7, Tp55, T14

Extended validation for CD27 Antibody, anti-human/mouse, 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 REA499
57703-
L128+
LG.3A10++
LG.7F9 (h/m/r)++
M-T271+
O323-
Cells were incubated with an excess of purified unconjugated CD27 (REA499) 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 CD27 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD27-PE (REA499, red) and counterstained with DRAQ5 (blue) as DNA stain.
View details
Fluorescence microscopy image of CD27 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD27-PE (REA499, red) and counterstained with DRAQ5 (blue) as DNA stain.
Fluorescence microscopy image of CD27 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD27-PE (REA499, red) and counterstained with DRAQ5 (blue) as DNA stain.
View details
Overlay histogram showing flow cytometric analysis of CD27 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD27-PE, clone (REA499). 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 CD27 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD27-PE, clone (REA499). 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 CD27. Human peripheral blood mononuclear cells (PBMCs) were stained with CD27antibodies and with a suitable counterstaining. As a control, CD27 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 CD27. Human peripheral blood mononuclear cells (PBMCs) were stained with CD27antibodies and with a suitable counterstaining. As a control, CD27 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 CD27. Human peripheral blood mononuclear cells (PBMCs) were stained with CD27antibodies and with a suitable counterstaining. As a control, CD27 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 CD27. Human peripheral blood mononuclear cells (PBMCs) were stained with CD27antibodies and with a suitable counterstaining. As a control, CD27 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 CD27. Human peripheral blood mononuclear cells (PBMCs) were stained with CD27antibodies and with a suitable counterstaining. As a control, CD27 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 CD27. Human peripheral blood mononuclear cells (PBMCs) were stained with CD27antibodies and with a suitable counterstaining. As a control, CD27 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 CD27. Human peripheral blood mononuclear cells (PBMCs) were stained with CD27antibodies and with a suitable counterstaining. As a control, CD27 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 CD27. Human peripheral blood mononuclear cells (PBMCs) were stained with CD27antibodies and with a suitable counterstaining. As a control, CD27 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 CD27 (REA499). 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 CD27 (REA499). 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 CD27 (REA499). 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 CD27 Antibody, anti-human/mouse, REAfinity™

Overview

Clone REA499 recognizes the human and mouse CD27 antigen, a single-pass type I membrane protein, also known as tumor necrosis factor receptor superfamily member 7 (TNFRSF7). CD27 is a member of the nerve growth factor receptor family and is exclusively expressed on cells of the lymphoid lineage, mostly in an activation-specific manner, and all enhance T cell receptor (TCR)–induced T cell expansion. CD27 and its ligand, CD70, have been defined at the protein, cDNA, and genomic level in both human and mouse. It is found on natural killer (NK), T, and B cell populations. In human and mice, the majority of CD4
+
and CD8
+
naive peripheral T cells express CD27 and expression is up-regulated upon TCR stimulation. Loss of CD27 expression is irreversible and seems to represent terminal effector T cell differentiation. In humans, naive B cells lack CD27 but antigen receptor stimulation induces expression. CD27
+
B cells display all the functional and phenotypic characteristics of memory cells.
Additional information: Clone REA499 displays negligible binding to Fc receptors.

Alternative names

S152, TNFRSF7, Tp55, T14

Detailed product information

Technical specifications

CloneREA499
Clonalitymonoclonal
Isotyperecombinant human IgG1
Isotype controlREA Control Antibody (S), human IgG1
Hostcell line
Type of antibodyPrimary antibodies, Recombinant antibodies
Specieshuman, mouse
AntigenCD27
Alternative names of antigenS152, TNFRSF7, Tp55, T14
Molecular mass of antigen [kDa]27, 26(separately shown for human and mouse/rat)
Distribution of antigenB cells, NK cells, T cells, thymocytes
Entrez Gene ID939, 21940
RRIDAB_2726193, AB_2726471, AB_2726194, AB_2751227, AB_2751162, AB_2751229, AB_2751164, AB_2819371, AB_2819368, AB_2751228, AB_2751163, AB_2801750, AB_2801743, AB_2811329, AB_2811328, AB_2752002, AB_2751971, AB_2811442, AB_2811436, AB_2801876, AB_2726470

Resources for CD27 Antibody, anti-human/mouse, REAfinity™

References for CD27 Antibody, anti-human/mouse, REAfinity™

Publications

  1. Camerini, D. et al. (1991) The T cell activation antigen CD27 is a member of the nerve growth factor/tumor necrosis factor receptor gene family. J. Immunol. 147(9): 3165-3169
  2. Prasad, K. V. et al. (1997) CD27, a member of the tumor necrosis factor receptor family, induces apoptosis and binds to Siva, a proapoptotic protein. Proc. Natl. Acad. Sci. U.S.A. 94(12): 6346-6351
  3. Gravestein, L. A. et al. (1993) Cloning and expression of murine CD27: comparison with 4-1BB, another lymphocyte-specific member of the nerve growth factor receptor family. Eur. J. Immunol. 23(4): 943-950

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