Skip to main content
Skip to main navigation

EuroMAbNet: European Monoclonal Antibodies Network

Antibody Identity card

Antibody information [MNDA]

253A

Clone name 253A
Description Mouse monoclonal
Antigen used Full length MNDA-GST recombinant protein
Epitope Unknown
Isotype IgG1
Confirmed species reactivity Human
Ab used Anti-MNDA antibody [253A] Abcam (ab188566) 1mg/ml

APPLICATION	Recommended concentration	Status	Protocol
Western blotting (WB)	10 ug/ml	Working	Western Blotting (WB)
Immunocytochemistry (ICC)	10 ug/ml	Working	ICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)	10 ug/ml	Working	BOND-MAX automated immunohistochemistry
Immunoflourescence (IF)	20 ug/ml	Working	Immunofluorescence staining
Flow cytometry (FC)	1ug/ml	Working	Flow cytometry
IHC-P Species		Not tested

Ab ID Card application validation / characterisation

WB Validation

Over-expression/cross reactivity

MNDA antibody WB Validation: Over-expression/cross reactivity — Western blotting (WB) of the 253A mAb using cell extracts of HEK293T cells transfected with V5-tagged human MNDA (HEK-MNDA-V5) as a positive control or MYC-tagged TOX (HEK-TOX-MYC) as a negative control. A 50 kDa band was present only in the HEK-MNDA-V5 cells. Anti-vinculin was used as a loading control.

Show image in a new window

Gene inactivation

MNDA antibody WB Validation: Gene inactivation — The specificity of the 253A mAb for the endogenous MNDA protein was confirmed by WB using cell extracts of the THP1 (acute monocytic leukaemia) cell line before (THP1) and after MNDA gene inactivation (MNDA_KO THP1) using CRISPR-Cas9 technology. Anti-vinculin was used as loading control.

Show image in a new window

WB Characterisation

Endogenous expression

MNDA antibody WB Characterisation: Endogenous expression — WB using the 253A mAb showed 50 kDa bands of MNDA protein in NB4 (acute promyelocytic leukaemia),THP1 (monocytic leukaemia) and U937 (myeloid leukaemia) cell lines. MNDA was not expressed in the RPMI8226 (plasmacytoma) and K562 (myeloid leukaemia) cell lines. Anti-LSD1 was used as a loading control.

Show image in a new window

ICC / ICC-P Validation

Over-expression/cross reactivity

MNDA antibody ICC Validation: Over-expression/cross reactivity — Immunocytochemistry (ICC) of the 253A mAb was performed on frozen cytospin preparations of HEK293T cells transfected with V5-tagged human MNDA or human CD4 proteins. Staining was observed only in the HEK-MNDA-V5 transfectants. Labelling with anti-V5 was used to confirm the efficiency of transfection.

Show image in a new window

Gene inactivation

MNDA antibody ICC Validation: Gene inactivation — The specificity of the 253A mAb for the endogenous MNDA protein was confirmed by ICC-P in sections from formalin fixed paraffin embedded pellets of the THP1 cell line before (THP1 WT) and after (MNDA_KO THP1) gene inactivation using CRISPR-Cas9 technology. MNDA expression was observed in the wild type (THP1 WT) cells, while no staining was observed in the MNDA_KO THP1 cells.

Show image in a new window

IHC-P Characterisation

Endogenous expression

MNDA antibody IHC-P Characterisation: Endogenous expression — Single immunoperoxidase of lymphoid tissues with the 253A mAb showed nuclear labelling of cells in the mantle zone of normal tonsil and lymphoid node. MNDA was also detected in the marginal and mantle zones of spleen. Germinal centres, monocytoid B cells and plasma cells were negative for MNDA. The 253A Mab also demonstrated strong nuclear labelling of mature granulocytes and monocytes in all the tissue samples studied.

Show image in a new window

IF Characterisation

Endogenous expression

MNDA antibody IF Characterisation: Endogenous expression — Triple immunofluorescence staining was performed on human paraffin-embedded spleen tissue using anti-TACI (clone CLOE204B, green), anti-CD20 (red) and anti-MNDA (clone 253C, white). Nuclei are stained by DAPI (blue). Weak MNDA expression was observed in B cells of the spleen marginal zone (white arrow), while strong MNDA staining was detected in the granulocytes (red arrow).

Show image in a new window

FC Validation

Over-expression/cross reactivity

MNDA antibody FC Validation: Over-expression/cross reactivity — Overexpression of MNDA protein was demonstrated using mAb 253A in flow cytometry. HEK293T cells transfected with MNDA were used as a positive control (light blue) while HEK293T cell transfected with TNIF1 were used as a negative control (pink). Control experiments using only the secondary antibody show no positive staining for HEK TNIF1 cells (dashed pink line) and HEK MNDA cells (dashed blue line).

Show image in a new window

Gene inactivation

MNDA antibody FC Validation: Gene inactivation — The specificity of the 253A mAb for the endogenous MNDA protein was confirmed by flow cytometry using THP1 cell line before (THP1 light blue) and after MNDA gene inactivation (THP1 MNDA KO, pink) using CRISPR-Cas9 technology. Control experiments using only the secondary antibody show no staining for THP1 MNDA KO cells (dashed pink line) and THP1 cells (dashed light blue line).

Show image in a new window

FC Characterisation

Endogenous expression

MNDA antibody FC Characterisation: Endogenous expression — Flow cytometry with the 253A mAb show endogenous MNDA protein expression in NB4 cells ( light blue ) but not in RPMI8226 cells (pink). Control experiments using only the secondary antibody show no staining for RPMI8226 cells (dashed pink line) and NB4 cells (dashed light blue line).

Show image in a new window

IHC-P Domestic species

Not tested

IHC-P Wild species

Not tested

← Back to MNDA protein