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Antibody Identity card

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

EPR23436-45

  • Clone name EPR23436-45
  • Description Rabbit monoclonal
  • Antigen used Synthetic peptide (information not available)
  • Epitope Unknown
  • Isotype IgG
  • Confirmed species reactivity Human
  • Ab used Abcam anti-AICDA mAb (Cat ab269454) 0,526mg/ml
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)1.052 μg/mlWorkingWestern Blotting (WB)
Immunocytochemistry (ICC)0.526 μg/mlWorkingICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)0.526 μg/mlWorkingBOND-MAX automated immunohistochemistry
Immunoflourescence (IF)1.315 μg/mlWorkingImmunofluorescence staining
Flow cytometry (FC)1.052 μg/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 EPR23436-45 mAb using cell extracts of HEK293T AID (HA-tagged) as positive control and HEK293T-hTDP2 (YFP-tagged) as negative control. A 25 kDa band was present only in the HEK293T AID cells. Anti-vinculin was used as loading control.

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Gene inactivation
WB Validation: Gene inactivation
The specificity of the EPR23436-45 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 only in the RAMOS WT cells. Anti-vinculin was used as loading control.

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WB Characterisation
Endogenous expression
WB Characterisation: Endogenous expression
WB to show 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 the wild type RAMOS, RAMOS activated with IL4+CD40, wild type BL-2, BL2 activated with IL4+CD40 and L428 cell lines. No band was observed in the U266 and HL-60 cell lines.

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ICC / ICC-P Validation
Over-expression/cross reactivity
ICC Validation: Over-expression/cross reactivity
Immunocytochemistry (ICC) to confirm the specificity of the EPR23436-45 mAb was performed on frozen cytospin preparations of HA-tagged human AID and FLAG-tagged TET1 proteins expressed in HEK293T cells. EPR23436-45 mAb stained only the HEK-AID-HA transfectants. Labelling with 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 EPR23436-45 mAb for endogenous AID protein was confirmed by ICC on sections of paraffin-fixed RAMOS cell line before (RAMOS WT) and after AID gene inactivation (RAMOS KO AID) using CRISPR-Cas9 technology. AID cytoplasmic expression was observed in RAMOS WT cells, while no staining was observed in RAMOS KO AID cells.

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IHC-P Characterisation
Endogenous expression
IHC-P Characterisation: Endogenous expression
In tonsil, EPR23436-4 mAb labelling showed AID to be highly expressed in germinal centre (GC) lymphocytes. This mAb shows some nonspecific staining in the tonsil epithelium (red arrow). 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 EPR23436-45 mAb (red) in human paraffin embedded tonsil showing AID expression in germinal centre cells.

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FC Validation
Over-expression/cross reactivity
FC Validation: Over-expression/cross reactivity
Overexpression of AID protein was demonstrated using the EPR23436-45 mAb in flow cytometry. HEK293T cells transfected with AID were used as a positive control (red) while HEK293T cell transfected with LCOR were used as a negative control (blue).

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Gene inactivation
FC Validation: Gene inactivation
Flow cytometry using the EPR23436-45 mAb showed endogenous AID expression in wild type RAMOS cells (blue) but not in the RAMOS AID KO cells (red).

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FC Characterisation
Endogenous expression
FC Characterisation: Endogenous expression
Endogenous expression of AID protein was demonstrated using the EPR23436-45 mAb in flow cytometry on the L428 cell line (blue). U266 cells (red) were used as a negative control.

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