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Antibody Identity card

Antibody information [MNDA]

EPR28267-44

  • Clone name EPR28267-44
  • Description Recombinant rabbit monoclonal
  • Antigen used The exact immunogen used to generate this antibody is proprietary information
  • Epitope Information not available
  • Isotype IgG
  • Confirmed species reactivity Human
  • Ab used Anti-MNDA antibody [EPR28267-44] abcam (0.51mg/ml)
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)5.1 ug/mlWorkingWestern Blotting (WB)
Immunocytochemistry (ICC)5.1 ug/mlWorkingICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)5.1 ug/mlWorkingBOND-MAX automated immunohistochemistry
Immunoflourescence (IF)10.2 ug/mlWorkingImmunofluorescence staining
Flow cytometry (FC)0.255 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 EPR28267-44 mAb using cell extracts of HEK293T transfected with either MNDA (HEK-MNDA-V5) as positive control or TOX2 (HEK-TOX2-MYC) as negative control. Two 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 EPR28267-44 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 EPR28267-44 mAb showed 50 kDa bands of MNDA protein in the NB4 (acute promyelocyic leukaemia) cell line. MNDA was not expressed in the U266 (multiple myeloma) cell line. Anti-vinculin 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 EPR28267-44 mAb was performed on frozen cytospin preparations of HEK293T cells transfected with V5-tagged human MNDA or mp15 proteins. Staining was observed only in the MNDA 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 EPR28267-44 mAb for the endogenous MNDA protein was confirmed by immunocytochemisty (ICC-P) in sections from formalin fixed paraffin embedded pellets of the THP1 cell line before 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 IHC-P immunoperoxidase staining with the EPR28267-44 mAb showed strong labelling of the nuclei of mature granulocyte and monocyte cells of all the tissue samples. Weaker labelling was also observed in the mantle zone cells of tonsil. In reactive lymph nodes, as well as in spleen tissue, the expression of MNDA is also detected in the marginal zone. The GCs of all follicles were negative for MNDA. Reactive monocytoid B cells and plasma cells were unlabelled.

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IF Characterisation
Endogenous expression
MNDA antibody IF Characterisation: Endogenous expression
Double immunofluorescence staining was performed on human paraffin-embedded spleen tissue using anti-CD20 (red) and anti-MNDA (clone EPR28267-44, green). 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 the MNDA protein was confirmed by flow cytometry using the mAb EPR28267-44. HEK293T cells transfected with MNDA served as a positive control (light blue), while HEK293T cells transfected with TNIF1 were used as a negative control (pink). Control experiments using only the secondary antibody showed no staining for HEK TNIF1 cells (dashed pink line) or HEK MNDA cells (dashed blue line).

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Gene inactivation
MNDA antibody FC Validation: Gene inactivation
The specificity of the EPR28267-44 mAb for endogenous MNDA protein was confirmed by flow cytometry using the THP1 cell line before (THP1, light blue) and after MNDA gene inactivation (THP1 MNDA KO, pink) via CRISPR-Cas9. Control experiments using only the secondary antibody showed no staining for THP1 cells (dashed light blue line) or THP1 MNDA KO cells (dashed pink line).

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FC Characterisation
Endogenous expression
MNDA antibody FC Characterisation: Endogenous expression
Flow cytometry using the EPR28267-44 mAb demonstrated endogenous MNDA protein expression in NB4 cells (light blue) but not in RPMI8226 cells (pink). Control experiments using only the secondary antibody showed no staining for RPMI8226 cells (dashed pink line) or 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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