Clone:
REA1023
Type of antibody:
Primary antibodies, Recombinant antibodies
Isotype:
recombinant human IgG1
Applications:
FC, MICS, IF, IHC
Alternative names:
LCA, L-CA, T200

Extended validation for CD45 Antibody, anti-non-human primate, 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 REA1023
5B1-
REA747-
2D1-
HI30-
REAL258-
MB4-6D6++
Cells were incubated with an excess of purified unconjugated CD45 (REA1023) 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 CD45 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD45-PE (REA1023, red) and counterstained with DRAQ5 (blue) as DNA stain.
View details
Fluorescence microscopy image of CD45 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD45-PE (REA1023, red) and counterstained with DRAQ5 (blue) as DNA stain.
Fluorescence microscopy image of CD45 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD45-PE (REA1023, red) and counterstained with DRAQ5 (blue) as DNA stain.
View details
Overlay histogram showing flow cytometric analysis of human CD45 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD45-PE, clone (REA1023). 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 human CD45 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD45-PE, clone (REA1023). 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 CD45. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45 antibodies and plotted against the side scatter. As a control, CD45 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 propidium iodide 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 CD45. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45 antibodies and plotted against the side scatter. As a control, CD45 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 propidium iodide 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 CD45. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45 antibodies and plotted against the side scatter. As a control, CD45 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 propidium iodide 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 CD45. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45 antibodies and plotted against the side scatter. As a control, CD45 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 propidium iodide 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 CD45. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45 antibodies and plotted against the side scatter. As a control, CD45 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 propidium iodide 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 CD45. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45 antibodies and plotted against the side scatter. As a control, CD45 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 propidium iodide 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 CD45. Human peripheral blood mononuclear cells (PBMCs) were stained with CD45 antibodies and plotted against the side scatter. As a control, CD45 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 propidium iodide 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 CD45 (REA1023). 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 CD45 (REA1023). 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 CD45 (REA1023). 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 CD45 Antibody, anti-non-human primate, REAfinity™

Overview

Clone REA1023 recognizes the rhesus monkey (
Macaca mulatta
) and cynomolgus monkey (
Macaca fascicularis
) CD45 antigen. It is reported to cross-react with pigtail monkey (
Macaca nemestrina
) and human. CD45 is a type I membrane glycoprotein also known as leukocyte common antigen (LCA), T200, or receptor-type tyrosine-protein phosphatase C (PTPRC). It is expressed on non-human primate hematopietic cells, such as lymphocytes, monocytes, and granulocytes. It is not found on erythroid cells. CD45 functions as a regulator for cell growth, differentiation, cell cycle, and oncogenic transformation.
Additional information: Clone REA1023 displays negligible binding to Fc receptors.

Alternative names

LCA, L-CA, T200

Detailed product information

Technical specifications

CloneREA1023
Clonalitymonoclonal
Isotyperecombinant human IgG1
Isotype controlREA Control Antibody (S), human IgG1
Hostcell line
Type of antibodyPrimary antibodies, Recombinant antibodies
Speciesnon-human primate, human
Cross-reactivity
rhesus monkey (
Macaca mulatta
)
,
cynomolgus monkey (
Macaca fascicularis
)
, human,
pigtail monkey (
Macaca nemestrina
)
AntigenCD45
Alternative names of antigenLCA, L-CA, T200
Molecular mass of antigen [kDa]41
Distribution of antigengranulocytes, lymphocytes, monocytes, leukocytes
RRIDAB_2727862, AB_2727900, AB_2727863, AB_2727901, AB_2727864, AB_2727902, AB_2727865, AB_2727903, AB_2727866, AB_2727904, AB_2727867, AB_2727905, AB_2727868, AB_2727869, AB_2727906, AB_2727870, AB_2727899

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