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Antibody Identity card

Antibody information [MNDA (Myeloid Cell Nuclear Differentiation Antigen)]

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
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)10 ug/mlWorkingWestern Blotting (WB)
Immunocytochemistry (ICC)10 ug/mlWorkingICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)10 ug/mlWorkingBOND-MAX automated immunohistochemistry
Immunoflourescence (IF)20 ug/mlWorkingImmunofluorescence staining
Flow cytometry (FC)1ug/mlWorkingFlow cytometry
IHC-P SpeciesNot tested

Ab ID Card application validation / characterisation

WB Validation
Over-expression/cross reactivity
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.

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Gene inactivation
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.

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WB Characterisation
Endogenous expression
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.

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ICC / ICC-P Validation
Over-expression/cross reactivity
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.

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Gene inactivation
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.

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IHC-P Characterisation
Endogenous expression
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.

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IF Characterisation
Endogenous expression
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).

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FC Validation
Over-expression/cross reactivity
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 (discontinuous pink line) and HEK MNDA cells (discontinuous blue line).

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Gene inactivation
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 (discontinuous pink line) and THP1 cells (discontinuous light blue line).

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FC Characterisation
Endogenous expression
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 (discontinuous pink line) and NB4 cells (discontinuous light blue line).

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IHC-P Domestic species
Not tested
IHC-P Wild species
Not tested
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