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Antibody Identity card

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

5G9

  • Clone name 5G9
  • Description Rat monoclonal
  • Antigen used Synthetic peptide within human AICDA (information not available)
  • Epitope Unknown
  • Isotype IgG2b
  • Confirmed species reactivity Human
  • Ab used Abcam (Cat ab252813) 1.264 mg/ml
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)2.528 μg/mlWorkingWestern Blotting (WB)
Immunocytochemistry (ICC)1.264 μg/mlWorkingICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)1.264 μg/mlWorkingBOND-MAX automated immunohistochemistry
Immunoflourescence (IF)3.16μg/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 5G9 mAb using cell extracts of HEK293T AID (HA-tagged) as positive control with HEK293T-hTDP2 (YFP-tagged) as negative control. A 25 kDa band was present only in the HEK293T AID cells. Anti-vinculin was used as a loading control.

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Gene inactivation
WB Validation: Gene inactivation
The specificity of the 5G9 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 a loading control.

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WB Characterisation
Endogenous expression
WB Characterisation: Endogenous expression
WB with the 5G9 mAb 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 IL-4+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. Anti-vinculin was used as a loading control.

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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 5G9 mAb was performed on frozen cytospin preparations of HA-tagged human AID or FLAG-tagged TET1 proteins expressed in HEK293T cells. 5G9 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 5G9 mAb for endogenous AID protein was confirmed by ICC on sections of the 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 the RAMOS WT cells, while no staining was observed in RAMOS KO AID.

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IHC-P Characterisation
Endogenous expression
IHC-P Characterisation: Endogenous expression
In tonsil, the 5G9 mAb labelling showed AID was 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 clone 5G9 mAB (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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