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Antibody Identity card

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

JUA51E

  • Clone name JUA51E
  • Description Mouse monoclonal
  • Antigen used EVDDLRDAFRMLGF peptide
  • Epitope EVDDLRDAFRMLGF
  • Isotype IgG1
  • Confirmed species reactivity Human
  • Ab used Anti-AID JUA51E (CNIO) tissue culture supernatant
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)Neat (tissue culture supernatant)WorkingWestern Blotting (WB)
Immunocytochemistry (ICC)Neat (tissue culture supernatant)WorkingICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)Neat (tissue culture supernatant)WorkingBOND-MAX automated immunohistochemistry
Immunoflourescence (IF)Neat (tissue culture supernatant)WorkingImmunofluorescence 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 JUA51E 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 JUA51E 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 cell extract. 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 wild type RAMOS, RAMOS activated with IL4+CD40, wild type BL-2 (weak band), BL-2 activated with IL4+CD40and L428 cell lines. No band was observed in the 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 JUA51E mAb. Labelling with the anti-HA and anti-FLAG mAbs was used to confirm the transfection efficiency.

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Gene inactivation
ICC Validation: Gene inactivation
The specificity of JUA51E mAb for the 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 wild type RAMOS WT cells, while no staining was observed in RAMOS KO AID transfectants.

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IHC-P Characterisation
Endogenous expression
IHC-P Characterisation: Endogenous expression
In tonsil, JUA51E mAb showed AID to be 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 clone JUA51E (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
Not tested
FC Characterisation
Endogenous expression
Not tested
IHC-P Domestic species
Not tested
IHC-P Wild species
Not tested
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