CD74 Antibody, anti-human, REAfinity™

Name: CD74 Antibody, anti-human, REAfinity™
Availability: InStock

CD74 Antibody, anti-human, REAfinity™Extended ValidationRecombinant

Clone:

REA1103

Type of antibody:

Primary antibodies, Recombinant antibodies

Isotype:

recombinant human IgG1

Applications:

FC, MICS, IF, IHC

Alternative names:

DHLAG, HLADG, II, Ia-GAMMA

Flow cytometry applications forCD74 Antibody, anti-human, REAfinity™

APC
APC-Vio 770
Biotin
PE
PE-Vio 615
PE-Vio 770
PerCP-Vio 700
Vio Bright B515
Vio Bright V423

Conjugate:

APC

Recommended dilution:

1:50

Remark:

The antibody is suited for staining of formaldehyde-fixed cells.

Tested:

QC tested

Products used:

130-119-204, 130-119-027

Protocols:

Cell surface flow cytometry staining protocol (PBS/EDTA/BSA 1:50)

Description:

Human peripheral blood mononuclear cells (PBMCs) were stained with CD74 antibodies or with the corresponding REA Control (S) antibodies (left images) as well as with CD19 antibodies and analyzed by flow cytometry using the MACSQuant^® Analyzer. 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 forCD74 Antibody, anti-human, REAfinity™

APC
PE

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

PFA

Tested:

during development

Species tested:

human

Products used:

130-119-204, 130-119-027

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

Acetone

Tested:

during development

Species tested:

human

Products used:

130-119-204, 130-119-027

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

Extended validation for CD74 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 REA1103
5-329	++
LN2	-
MB741	+
Cells were incubated with an excess of purified unconjugated CD74 (REA1103) 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

Fluorescence microscopy image of CD74 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD74-PE (REA1103, red) and counterstained with DRAQ5 (blue) as DNA stain.

Fluorescence microscopy image of CD74 knockout cells. Wild type (WT, left) and knockout cells (KO, right) were stained with CD74-PE (REA1103, red) and counterstained with DRAQ5 (blue) as DNA stain.

Overlay histogram showing flow cytometric analysis of human CD74 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD74-PE, clone (REA1103). 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 CD74 knockout cells. Wild type (red) and knockout cells (blue) were stained with CD74-PE, clone (REA1103). 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.

Flow cytometric comparison of different clones for CD74. Human peripheral blood mononuclear cells (PBMCs) were stained with CD74 antibodies and with a suitable counterstaining. As a control, CD74 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 CD74. Human peripheral blood mononuclear cells (PBMCs) were stained with CD74 antibodies and with a suitable counterstaining. As a control, CD74 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 CD74. Human peripheral blood mononuclear cells (PBMCs) were stained with CD74 antibodies and with a suitable counterstaining. As a control, CD74 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 CD74 (REA1103). 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 CD74 (REA1103). 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 CD74 Antibody, anti-human, REAfinity™

Overview

Clone REA1103 recognizes CD74, a non-polymorphic type II integral membrane protein. CD74 is also known as MHC class II–associated invariant chain and exists in multiple isoforms in human ranging from 33 to 43 kDa in size. Initially known for its role as the MHC II chaperone, CD74 primary function is suggested to regulate the MHC class II molecules folding and intracellular sorting. CD74 is also shown to form complexes with CD182 (CXCR2) and CD184 (CXCR4). A direct role of CD74 in the maturation of follicular B cells and accumulation of marginal zone B cells has also been suggested. CD74 was further shown to be one of the host molecules utilized by

H. pylori

to successfully attach to the gastric epithelial cells. Expression of CD74 is found on B cells, dendritic cells, epithelial cells, subsets of activated T cells, and low level expression is found on monocytes and macrophages.

Additional information: Clone REA1103 displays negligible binding to Fc receptors.

Alternative names

DHLAG, HLADG, II, Ia-GAMMA

Detailed product information

Technical specifications

Clone	REA1103
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	CD74
Alternative names of antigen	DHLAG, HLADG, II, Ia-GAMMA
Molecular mass of antigen [kDa]	33
Distribution of antigen	B cells, dendritic cells, epithelial cells, macrophages, monocytes, T cells
Entrez Gene ID	972
RRID	AB_2733836, AB_2733837, AB_2733403, AB_2733404, AB_2732984, AB_2732985, AB_2733248, AB_2733249, AB_2733143, AB_2733144, AB_2733041, AB_2733042, AB_2734027, AB_2734028, AB_2733534, AB_2733535, AB_2921918, AB_2921887

Resources for CD74 Antibody, anti-human, REAfinity™

Certificates

Please follow this

link

to search for Certificates of Analysis (CoA) by lot number.

References for CD74 Antibody, anti-human, REAfinity™

Publications

Leng, L. et al. (2003) MIF signal transduction initiated by binding to CD74. J. Exp. Med. 197: 1467-1476
Beswick, E. J. and Reyes, V. E. (2009) CD74 in antigen presentation, inflammation, and cancers of the gastrointestinal tract. World J Gastroenterol. 15(23): 2855-2861

Applications

Extended validation