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Antibody Identity card

Antibody information [AID (Activation-induced cytidine deaminase) ]

mAID-2

  • Clone name mAID-2
  • Description Rat monoclonal
  • Antigen used Synthetic peptide corresponding to Mouse AICDA (C terminal) conjugated to keyhole limpet haemocyanin.
  • Epitope Unknown
  • Isotype IgG2a
  • Confirmed species reactivity Human
  • Ab used Abcam mAID-2 ab169522 (0.5 mg/ml)
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)6.25 μg/mlWorkingWestern Blotting (WB)
Immunocytochemistry (ICC)5 μg/mlWorkingICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)5 μg/mlWorkingBOND-MAX automated immunohistochemistry
Immunoflourescence (IF)10 μg/mlWorkingImmunofluorescence staining
Flow cytometry (FC)Not tested
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 mAID-2 mAb using cell extracts of HEK293T AID (HA-tagged) as positive control and HEK293T-hTDP2 (YFP-tagged) as negative control. m-AID2 stained only the HEK293T AID HA transfectants. Anti-vinculin was used as loading control

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Gene inactivation
WB Validation: Gene inactivation
The specificity of the mAID-2 mAb for the endogenous AID protein was confirmed by WB using cell extracts of the RAMOS cell line before (RAMOS WT) and after AID gene inactivation (RAMOS AID KO) using CRISPR-Cas9 technology. A 25 kDA band was present in the RAMOS WT cells extracts only. Anti-vinculin was used as loading control.

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WB Characterisation
Endogenous expression
WB Characterisation: Endogenous expression
WB to demonstrate endogenous expression of AID protein in the following cell lines: wild type RAMOS, RAMOS activated with IL4+CD40, wild type BL-2, BL-2 activated with IL4+CD40, L428, U266 and HL-60. As expected, a specific 25kDa band was observed in wild type RAMOS WT, RAMOS activated with IL4+CD40, BL-2 activated with IL4+CD40 and L428 cell lines. No band was observed in wild type BL-2, U266 and HL-60 cell lines. Anti-vinculin was used as loading control.

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ICC / ICC-P Validation
Over-expression/cross reactivity
ICC Validation: Over-expression/cross reactivity
Immunocytochemistry (ICC) was performed on frozen cytospin preparations of HA-tagged human AID and FLAG-tagged TET1 proteins expressed in HEK293T cells to confirm the specificity of the mAID-2 mAb. m-AID-2 stained only the HEK 293T mAID-2 HA transfectants. Labelling using the anti-HA and anti-FLAG were used to confirm the efficiency of transfection.

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Gene inactivation
ICC Validation: Gene inactivation
The specificity of the mAID-2 mAb for the endogenous AID protein was confirmed by ICC on paraffin embedded sections of the RAMOS cell line before (RAMOS WT) and after AID gene inactivation (RAMOS KO AID) using CRISPR-Cas9 technology. Cytoplasmic expression was observed in the RAMOS WT cells, while no staining was observed in the RAMOS KO AID cells.

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IHC-P Characterisation
Endogenous expression
IHC-P Characterisation: Endogenous expression
In tonsil, the mAID-2 mAb showed AID to be highly expressed in germinal centre (GC) lymphocytes. Spleen with and without marginal zone hyperplasia contained AID-positive GC cells and scattered single cells in the marginal zone. Thymus was negative.

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IF Characterisation
Endogenous expression
IF Characterisation: Endogenous expression
Single immunofluorescence staining of the mAID-2mAb clone (red) in human paraffin embedded tonsil showing AID expression in germinal centres.

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FC Validation
Over-expression/cross reactivity
FC Validation: Over-expression/cross reactivity
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Gene inactivation
FC Validation: Gene inactivation
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FC Characterisation
Endogenous expression
Not tested
IHC-P Domestic species
Not tested
IHC-P Wild species
Not tested
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