Clone:
REA260
Type of antibody:
Primary antibodies, Recombinant antibodies
Isotype:
recombinant human IgG1
Applications:
FC, MICS, IF, IHC
Alternative names:
INSR, HHF5

Extended validation for CD220 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 REA260
3B6/IR++
B6.220++
Cells were incubated with an excess of purified unconjugated CD220 (REA260) 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.
View details
Overlay histogram showing flow cytometric analysis of CD220 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD220-PE, clone (REA260). 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 CD220 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD220-PE, clone (REA260). 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 CD220. Human peripheral blood mononuclear cells (PBMCs) were stained with CD220 antibodies and with a suitable counterstaining. As a control, CD220 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 CD220. Human peripheral blood mononuclear cells (PBMCs) were stained with CD220 antibodies and with a suitable counterstaining. As a control, CD220 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 CD220. Human peripheral blood mononuclear cells (PBMCs) were stained with CD220 antibodies and with a suitable counterstaining. As a control, CD220 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 CD220. Human peripheral blood mononuclear cells (PBMCs) were stained with CD220 antibodies and with a suitable counterstaining. As a control, CD220 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 CD220. Human peripheral blood mononuclear cells (PBMCs) were stained with CD220 antibodies and with a suitable counterstaining. As a control, CD220 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 CD220 (REA260). 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 CD220 (REA260). 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 CD220 (REA260). 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 CD220 Antibody, anti-human, REAfinity™

Overview

Clone REA260 recognizes the CD220 antigen, a type I transmembrane receptor tyrosine kinase, which is also known as insulin receptor (IR). CD220 is predominantly expressed in major target tissues of insulin metabolic effects like liver, adipose tissue, and skeletal muscle, but is also expressed in the peripheral nerve, kidney, fibroblasts, monocytes, granulocytes, erythrocytes, and skin. It is composed of two extracellular α-subunits and two transmembrane β-subunits. Upon binding of insulin, CD220 forms a heterotetramer of two units to induce autophosphorylation and activation of the tyrosine kinase activity of the receptor. CD220 plays a key role in the regulation of glucose homeostasis, cell growth, differentiation, and apoptosis- functional processes that under degenerate conditions may result in a range of clinical manifestations including diabetes and cancer.
Additional information: Clone REA260 displays negligible binding to Fc receptors.

Alternative names

INSR, HHF5

Detailed product information

Technical specifications

CloneREA260
Clonalitymonoclonal
Isotyperecombinant human IgG1
Isotype controlREA Control Antibody (S), human IgG1
Hostcell line
Type of antibodyPrimary antibodies, Recombinant antibodies
Specieshuman
AntigenCD220
Alternative names of antigenINSR, HHF5
Molecular mass of antigen [kDa]307(sum of molecular weights of subunits)
Distribution of antigenmonocytes, fibroblasts, red blood cells, granulocytes, liver
Entrez Gene ID3643
RRIDAB_2656379, AB_2656380, AB_2656381, AB_2656382, AB_2656383, AB_2656384

Resources for CD220 Antibody, anti-human, REAfinity™

Certificates

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

References for CD220 Antibody, anti-human, REAfinity™

Publications

  1. Youngren, J. F. et al. (2007) Regulation of insulin receptor function. Cell. Mol. Life Sci. 64(7-8): 873-891
  2. Czech, M. P. et al. (1985) The nature and regulation of the insulin receptor: structure and function. Annu. Rev. Physiol. 47: 357-381
  3. Chiu, S. L. et al. (2010) Insulin receptor signaling in the development of neuronal structure and function. Neural Dev. 5: 7

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