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Antibody Identity card

Antibody information [MNDA]

5C

  • Clone name 5C
  • Description Mouse monoclonal
  • Antigen used Full length MNDA-GST recombinant protein
  • Epitope Unknown
  • Isotype IgG1
  • Confirmed species reactivity Human
  • Ab used Purified antibody (5C) CNIO (2.27 mg/ml)
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)22.7 ug/mlWorkingWestern Blotting (WB)
Immunocytochemistry (ICC)5.7 ug/mlWorkingICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)22.7 ug/mlWorkingBOND-MAX automated immunohistochemistry
Immunoflourescence (IF)37.83 ug/mlWorkingImmunofluorescence staining
Flow cytometry (FC)2.27 ug/mlWorkingFlow cytometry
IHC-P SpeciesNot 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 5C mAb using cell extracts of HEK293T transfected with MNDA (HEK-MNDA-V5) as a positive control or TOX (HEK-TOX-MYC-tagged) as a negative control. Bands of 48 and 55 kDa bands were present only in the HEK293T-MNDA-V5 cells. Anti-vinculin was used as a loading control.

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Gene inactivation
MNDA antibody WB Validation: Gene inactivation
The specificity of the 5C mAb for the endogenous MNDA protein was confirmed by WB using cell extracts of THP1 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
MNDA antibody WB Characterisation: Endogenous expression
WB using the 5C mAb showed 50 kDa bands of MNDA protein in NB4 (acute promyelocytic leukemia), THP1 (acute monocytoid leukaemia) and U937 (myeloid leukaemia) cell lines. MNDA was not expressed in the RPMI8226 (multiple myeloma) and K562 (myeloid leukaemia) cell lines. LSD1 was used as a loading control.

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ICC / ICC-P Validation
Over-expression/cross reactivity
MNDA antibody ICC Validation: Over-expression/cross reactivity
Immunocytochemistry (ICC) of the 5C mAb was performed on frozen cytospin preparations of HEK293T cells transfected with either V5-tagged human MNDA or human CD4 proteins. Staining was observed only in the MNDA (HEK-MNDA-V5) transfectants. Labelling with anti-V5 was used to confirm the efficiency of transfection

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Gene inactivation
MNDA antibody ICC Validation: Gene inactivation
The specificity of the 5C mAb for the endogenous MNDA protein was confirmed by immunocytochemistry (ICC-P) in sections from fomalin fixed paraffin embedded cell pellets of the THP1 cell line before (THP1) and after (MNDA_KO THP1) gene inactivation using CRISPR-Cas9 technology. MNDA expression was observed in wild type THP1 cells, while no staining was observed in the MNDA_KO THP1 cells.

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IHC-P Characterisation
Endogenous expression
MNDA antibody IHC-P Characterisation: Endogenous expression
Single immunoperoxidase labelling of lymphoid tissues with the 5C mAb showed nuclear labelling of cells in the mantle zone of reactive tonsil. MNDA was also detected in the marginal and mantle zones of spleen. Germinal centres were negative for MNDA. MNDA is also expressed in the myeloid component in thymus and lung. The 5C 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
MNDA antibody IF Characterisation: Endogenous expression
Triple immunofluorescence staining was performed on human paraffin-embedded spleen tissue using anti-TACI Mab (clone CLOE204B, green), anti-CD20 (red), and anti-MNDA (clone 5C, white). Weak MNDA expression was observed in B cells of the spleen marginal zone, while strong MNDA staining was detected in the granulocytes.

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FC Validation
Over-expression/cross reactivity
MNDA antibody FC Validation: Over-expression/cross reactivity
Overexpression of MNDA protein was demonstrated using mAb 5C 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).

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Gene inactivation
MNDA antibody FC Validation: Gene inactivation
The specificity of the 5C 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).

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FC Characterisation
Endogenous expression
MNDA antibody FC Characterisation: Endogenous expression
Flow cytometry with the 5C 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).

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