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Antibody Identity card

Antibody information [CD229 (Lymphocyte Antigen 9, LY9)]

EPR22611-91

  • Clone name EPR22611-91
  • Description Rabbit monoclonal
  • Antigen used Recombinant fragment within human CD229 aa 1-500
  • Epitope Unknown
  • Isotype IgG
  • Confirmed species reactivity Human
  • Ab used Abcam (Cat. ab252931) 0.551 mg/ml
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)1.1 μg/mlWorkingOdyssey Western Blotting protocol (OdWB)
Immunocytochemistry (ICC)0.7 μg/mlWorkingICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)0.7 μg/mlWorkingBOND-MAX automated immunohistochemistry
Immunoflourescence (IF)1.8 μg/mlWorkingImmunofluorescence staining
Flow cytometry (FC)1.5 μg/mlNot workingFlow cytometry
IHC-P Species0.7 μg/mlNot workingBOND-MAX automated immunohistochemistry

Ab ID Card application validation / characterisation

WB Validation
Over-expression/cross reactivity
WB Validation: Over-expression/cross reactivity
Western blotting (WB) of the EPR22611-91 mAb using cell extracts of HEK293T hCD229/LY9 (GFP-tagged) as positive control and HEK293T-GCET2 as negative control. Bands of 75 to 100 kDa were observed only in the HEK293T hCD229/LY9 transfectants. Anti-GAPDH was used as loading control.

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Gene inactivation
WB Validation: Gene inactivation
The specificity of the EPR22611-91 mAb for the endogenous CD229/LY9 protein was confirmed by WB using cell extracts of the U266 cell line before and after LY9 gene inactivation (U266 KO LY9) using CRISPR-Cas9 technology. Three bands around 75-110kDa were detected in the U266 WT (corresponding with the three LY9 isoforms) cells while no bands were found in U266 KO LY9. Anti-GAPDH was used as loading control.

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WB Characterisation
Endogenous expression
WB Characterisation: Endogenous expression
The detection of the endogenous expression of CD229/LY9 protein using the EPR22611-91 mAb was studied by WB in DAUDI, SUDHL4 and L363 cell lines and human tonsil and liver tissues. Three bands corresponding to the three CD229/LY9 isoforms were detected in the DAUDI, SUDHL4, L-363 cell lines and human tonsil while human liver was negative. Anti-GAPDH 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) to confirm the specificity of the EPR22611-91 mAb was performed on frozen cytospin preparations of GFP-tagged human CD229/LY9 and MYC-tagged LILRB3 proteins expressed in HEK293T cells. Labelling with the EPR22611-54 mAb was present in the HEK-LY9-GFP transfectants but not in the negative control LILRB3-MYC cells. Labelling with anti-GFP and anti-MYC were used to confirm transfection efficiency.

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Gene inactivation
Not working
IHC-P Characterisation
Endogenous expression
IHC-P Characterisation: Endogenous expression
Immunohistochemical labelling with the EPR22611-91 mAb showed CD229/LY9 was expressed in haematopietic tissues. A) In tonsil, strong expression was detected in B- and T-cells in the germinal centre, mantle zone and interfollicular areas as well as plasma cells in the subepithelium and mantle zone. B) Mature T-cells in the thymic medulla were strongly positive with weaker expression in the thymic cortex. C) Strong expression was also detected in mantle and marginal zones as well as some T-cells within the white-pulp area of the spleen. D) Scattered cells were CD229/LY9-positive in bone marrow.

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IF Characterisation
Endogenous expression
IF Characterisation: Endogenous expression
Immunofluorescence staining of formalin fixed human tonsil with the EPR22611-91 mAb (green), anti-PD-1 (white) and anti-CD8 (red).

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