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Antibody Identity card

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

PIZCU426A

  • Clone name PIZCU426A
  • Description Rat monoclonal
  • Antigen used HIS-CD229 intracellular domain (C-term 477-655aa) recombinant protein
  • Epitope Unknown
  • Isotype IgG2a
  • Confirmed species reactivity Human
  • Ab used Ly9 CNIO (clone PIZCU426A) (3 mg/ml)
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)15 μg/mlWorkingOdyssey Western Blotting protocol (OdWB)
Immunocytochemistry (ICC)1 μg/mlWorkingICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)4.68 μg/mlWorkingBOND-MAX automated immunohistochemistry
Immunoflourescence (IF)10 μg/mlWorkingImmunofluorescence staining
Flow cytometry (FC)Not working
IHC-P Species4.68 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 PIZCU426A mAb using cell extracts of HEK293T hCD229/LY9 (GFP-tagged) as positive control and HEK293T-GCET2 as negative control. Three bands of 100 to 75 kDa were present 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 PIZCU426A mAb for the endogenous CD229/LY9 protein was confirmed by WB using two different cell extracts: U266 cell line (U266 WT) and U266 after LY9 gene inactivation (U266 KO LY9) using CRISPR-Cas9 technology. Three bands approximately 75-110 kDa were detected in U266 WT (corresponding with the three LY9 isoforms) and no band was found in the U266 CD229 KO LY9 extract. Anti-GAPDH was used as loading control.

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WB Characterisation
Endogenous expression
WB Characterisation: Endogenous expression
The detection of endogenous expression of CD229/LY9 protein using the PIZCU426A mAb was confirmed by WB using the DAUDI, SUDHL4 and L363 cell lines and human tonsil and liver. Extracts of DAUDI, SUDHL4 and human tonsil showed three bands corresponding to the three isoforms of CD229/LY9. No bands were observed in the L-363 cell line and human liver. 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 PIZCU426A mAb was performed on frozen cytospin preparations of GFP-tagged human CD229/LY9 and MYC-tagged LILRB3 proteins expressed in HEK293T cells. Staining was detected only in the CD229/LY9 transfectants. Labelling with anti-MYC and anti-GFP were used to confirm the efficiency of transfection.

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Gene inactivation
Not working
IHC-P Characterisation
Endogenous expression
IHC-P Characterisation: Endogenous expression
Immunohistochemical labelling with the OT12H4 mAb showed CD229/LY9 was strongly expressed in haematopietic tissues. A) In tonsil, strong expression was detected in B- and T-cells in the interfollicular areas and in plasma cells in the subepithelium outside the follicles as well as in the 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 spleen. D) Scattered cells were CD229/LY9-positive in bone marrow.

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IF Characterisation
Endogenous expression
IF Characterisation: Endogenous expression
A. Immunofluorescence (IF) in U266 cells shows triple staining of the PIZCU426A mAb (green), anti-clathrin (red) and anti-LAMP-2 (blue). B. IF of paraffin fixed human tonsil with the PIZCU426A mAb (red), anti-PD1 (green) and anti-CD20 (blue).

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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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