CD235a (Glycophorin A) Antibody, anti-human, REAfinity™

Name: CD235a (Glycophorin A) Antibody, anti-human, REAfinity™
Availability: InStock

CD235a (Glycophorin A) Antibody, anti-human, REAfinity™Extended ValidationRecombinant

Clone:

REA175

Type of antibody:

Primary antibodies, Recombinant antibodies

Isotype:

recombinant human IgG1

Applications:

FC, MICS, IF, IHC, MC

Alternative names:

GYPA, GPA, GPErik, GPSAT, HGpMiV, HGpMiXI, HGpSta(C), MN, MNS, PAS-2

Flow cytometry applications forCD235a (Glycophorin A) Antibody, anti-human, REAfinity™

APC
APC-Vio 770
Biotin
FITC
PE
PE-Vio 615
PE-Vio 770
PerCP-Vio 700
Vio Bright B515
VioBlue
VioGreen

Conjugate:

APC

Recommended dilution:

1:50

Remark:

Cells should be stained prior to fixation, if formaldehyde is used as a fixative.

Tested:

QC tested

Products used:

130-118-493, 130-118-356

Protocols:

Description:

Human peripheral blood cells were stained with CD235a (Glycophorin A) antibodies and analyzed by flow cytometry using the MACSQuant^® Analyzer. Pre-gating was performed on CD45^– single cells. Left peak represents unstained cells. The Tandem Signal Enhancer has been used to increase binding specificity of tandem-dye–conjugated antibodies. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence or 4',6-diamidino-2-phenylindole (DAPI) fluorescence, as in the case of tandem conjugates.

MICS (MACSima Imaging Cyclic Staining) applications forCD235a (Glycophorin A) Antibody, anti-human, REAfinity™

APC
FITC
PE

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

FFPE

Tested:

during development

Species tested:

human

Products used:

130-118-493, 130-118-356

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

PFA

Tested:

during development

Species tested:

human

Products used:

130-118-493, 130-118-356

Compatible applications:

IF, IHC, ICC

Protocols:

Protocol for MICS experiments

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

Acetone

Tested:

during development

Species tested:

human

Products used:

130-118-493, 130-118-356

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

Mass cytometry applications forCD235a (Glycophorin A) Antibody, anti-human, REAfinity™

pure

Conjugate:

pure

Products used:

130-108-036

Extended validation for CD235a (Glycophorin A) 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 clones	Overlap in epitope recognition with REA175
GA-RA (HIR2)	+
HI264	-
YTH89.1	-
10F7MN	-
CLB-ery-1 (AME-1)	-
Cells were incubated with an excess of purified unconjugated CD235a (Glycophorin A) (REA175) 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.

Sensitivity

Performance comparison

Selected fluorochrome conjugated antibodies from Miltenyi Biotec were compared to commercially available hybridoma clones in flow cytometry analysis.

Flow cytometric comparison of different clones for CD235a (Glycophorin A). A mixture of human peripheral blood mononuclear cells (PBMCs) and 2% whole blood were stained with CD235a (Glycophorin A) antibodies and plotted against the side scatter. As a control, CD235a (Glycophorin A) 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 CD235a (Glycophorin A). A mixture of human peripheral blood mononuclear cells (PBMCs) and 2% whole blood were stained with CD235a (Glycophorin A) antibodies and plotted against the side scatter. As a control, CD235a (Glycophorin A) 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 CD235a (Glycophorin A). A mixture of human peripheral blood mononuclear cells (PBMCs) and 2% whole blood were stained with CD235a (Glycophorin A) antibodies and plotted against the side scatter. As a control, CD235a (Glycophorin A) 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 CD235a (Glycophorin A). A mixture of human peripheral blood mononuclear cells (PBMCs) and 2% whole blood were stained with CD235a (Glycophorin A) antibodies and plotted against the side scatter. As a control, CD235a (Glycophorin A) 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 CD235a (Glycophorin A). A mixture of human peripheral blood mononuclear cells (PBMCs) and 2% whole blood were stained with CD235a (Glycophorin A) antibodies and plotted against the side scatter. As a control, CD235a (Glycophorin A) 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 CD235a (Glycophorin A). A mixture of human peripheral blood mononuclear cells (PBMCs) and 2% whole blood were stained with CD235a (Glycophorin A) antibodies and plotted against the side scatter. As a control, CD235a (Glycophorin A) 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 CD235a (Glycophorin A). A mixture of human peripheral blood mononuclear cells (PBMCs) and 2% whole blood were stained with CD235a (Glycophorin A) antibodies and plotted against the side scatter. As a control, CD235a (Glycophorin A) 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

Comparison of staining pattern on non-fixed and fixed cells using CD235a (Glycophorin A) (REA175). 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 CD235a (Glycophorin A) (REA175). 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 CD235a (Glycophorin A) Antibody, anti-human, REAfinity™

Overview

Clone REA175 recognizes CD235a, commonly known as glycophorin A. CD235a is a single-pass transmembrane glycoprotein and is expressed on mature erythrocytes and erythroid precursor cells. It is one of the major sialoglycoprotein expressed on human red blood cells.

The CD235a (Glycophorin A) antibody has been tested by flow cytometric analysis of human peripheral blood cells according to the protocol. Binding of the antibody to red cells at high and moderate antibody concentration can cause cell agglutination, so it is recommended to titrate the antibody carefully for optimal performance in the assay of interest.

Additional information: Clone REA175 displays negligible binding to Fc receptors.

Alternative names

GYPA, GPA, GPErik, GPSAT, HGpMiV, HGpMiXI, HGpSta(C), MN, MNS, PAS-2

Detailed product information

Technical specifications

Clone	REA175
Clonality	monoclonal
Isotype	recombinant human IgG1
Isotype control	REA Control Antibody (S), human IgG1
Host	human cell line
Type of antibody	Primary antibodies, Recombinant antibodies
Species	human
Antigen	CD235a (Glycophorin A)
Alternative names of antigen	GYPA, GPA, GPErik, GPSAT, HGpMiV, HGpMiXI, HGpSta(C), MN, MNS, PAS-2
Molecular mass of antigen [kDa]	14
Distribution of antigen	red blood cells
Entrez Gene ID	2993
RRID	AB_2728019, AB_2751527, AB_2751491, AB_2801777, AB_2801768, AB_2784083, AB_2784082, AB_2784085, AB_2784084, AB_2752159, AB_2752109, AB_2819396, AB_2819393, AB_2784081, AB_2784080, AB_2819397, AB_2819394, AB_2656511, AB_2921768, AB_2921767, AB_2928345, AB_2928344, AB_2728042

Resources for CD235a (Glycophorin A) Antibody, anti-human, REAfinity™

Documents and Protocols

Scientific posters

A novel user-independent CFU assay for hematopoietic stem and progenitor cells

Certificates

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References for CD235a (Glycophorin A) Antibody, anti-human, REAfinity™

Publications

Chasis, J. A. and Mohandas, N. (1992) Red blood cell glycophorins. Blood 80(8): 1869-1879

Applications

Extended validation