GARP (LRRC32) Antibody, anti-human, REAfinity™

Name: GARP (LRRC32) Antibody, anti-human, REAfinity™
Availability: InStock

GARP (LRRC32) Antibody, anti-human, REAfinity™Extended ValidationRecombinant

Clone:

REA166

Type of antibody:

Primary antibodies, Recombinant antibodies

Isotype:

recombinant human IgG1

Applications:

FC

Alternative names:

LRRC32, D11S833E, Glycoprotein A repetitions predominant (GARP)

Flow cytometry applications forGARP (LRRC32) Antibody, anti-human, REAfinity™

Biotin
PE
Vio Bright B515

Conjugate:

Biotin

Recommended dilution:

1:11

Remark:

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

Tested:

QC tested

Products used:

130-104-362, 130-104-313

Protocols:

Cell surface flow cytometry staining protocol (PBS/EDTA/BSA 1:11)

Description:

Human peripheral blood mononuclear cells (PBMCs), either left unstimulated (left images) or stimulated with CD3/CD28/IL-2 for 2 days, were surface-stained with Anti-GARP (LRRC32) antibodies. Cells were then fixed and permeabilized using the FoxP3 Staining Buffer Set followed by intracellular staining with Anti-FoxP3 antibodies and analyzed by flow cytometry using the MACSQuant^® Analyzer. CD4⁺ cells were pre-gated for the analysis. Cell debris were excluded from the analysis based on scatter signals.

Extended validation for GARP (LRRC32) 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 REA166
855151	-
7B11	++
G14D9	-
Cells were incubated with an excess of purified unconjugated GARP (LRRC32) (REA166) 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 Anti-GARP (LRRC32). Human peripheral blood mononuclear cells (PBMCs) stimulated with CD3/CD28 antibodies (T Cell TransAct™) for 24 hours were stained with Anti-GARP (LRRC32) antibodies and with a suitable counterstaining. As a control, Anti-GARP (LRRC32) antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^®Analyzer. CD4⁺ cells were pregated for the analysis. 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 Anti-GARP (LRRC32). Human peripheral blood mononuclear cells (PBMCs) stimulated with CD3/CD28 antibodies (T Cell TransAct™) for 24 hours were stained with Anti-GARP (LRRC32) antibodies and with a suitable counterstaining. As a control, Anti-GARP (LRRC32) antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^®Analyzer. CD4⁺ cells were pregated for the analysis. 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 Anti-GARP (LRRC32). Human peripheral blood mononuclear cells (PBMCs) stimulated with CD3/CD28 antibodies (T Cell TransAct™) for 24 hours were stained with Anti-GARP (LRRC32) antibodies and with a suitable counterstaining. As a control, Anti-GARP (LRRC32) antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^®Analyzer. CD4⁺ cells were pregated for the analysis. 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 Anti-GARP (LRRC32). Human peripheral blood mononuclear cells (PBMCs) stimulated with CD3/CD28 antibodies (T Cell TransAct™) for 24 hours were stained with Anti-GARP (LRRC32) antibodies and with a suitable counterstaining. As a control, Anti-GARP (LRRC32) antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^®Analyzer. CD4⁺ cells were pregated for the analysis. 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 GARP (LRRC32) (REA166). 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 GARP (LRRC32) (REA166). 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 GARP (LRRC32) Antibody, anti-human, REAfinity™

Overview

Clone REA166 recognizes the human glycoprotein A repetitions predominant (GARP) antigen, a 80 kDa type I membrane glycoprotein which belongs to the family of leucine rich repeat proteins. Expression of GARP protein is found on megakaryocytes, platelets, and activated regulatory T cells (Treg cells). GARP has been found to be associated with latency associated peptide (LAP), the prodomain responsible for conferring latency to non-active TGF-β. Thus, GARP as a latent TGF-β–binding protein (LTBP) regulates the surface expression of TGF-β and serves as a carrier of LAP, at the cell surface, to immune response active sites, where TGF-β is eventually activated and released to exert further physiological functions such as differentiation of regulatory T cells.

Additional information: Clone REA166 displays negligible binding to Fc receptors.

Alternative names

LRRC32, D11S833E, Glycoprotein A repetitions predominant (GARP)

Detailed product information

Technical specifications

Clone	REA166
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	GARP (LRRC32)
Alternative names of antigen	LRRC32, D11S833E, Glycoprotein A repetitions predominant (GARP)
Molecular mass of antigen [kDa]	70
Distribution of antigen	T cells, monocytes, platelets
Entrez Gene ID	2615
RRID	AB_2801741, AB_2651819, AB_2651820, AB_2905216, AB_2905215, AB_2801748

Resources for GARP (LRRC32) Antibody, anti-human, REAfinity™

Certificates

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References for GARP (LRRC32) Antibody, anti-human, REAfinity™

Publications

Zhou, A. X. et al. (2013)
GARP-TGF-β complexes negatively regulate regulatory T cell development and maintenance of peripheral CD4
⁺
T cells
in vivo
.
J Immunol 190(10): 5057-5064
Tran, D. Q. et al. (2009)
GARP (LRRC32) is essential for the surface expression of latent TGF-β on platelets and activated FOXP3
⁺
regulatory T cells.
Proc. Natl. Acad. Sci. U.S.A. 106: 13445-13450
Ollendorff, V. et al. (1994) The GARP gene encodes a new member of the family of leucine-rich repeat-containing proteins. Cell Growth Differ. 5: 213-219

Applications

Extended validation