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Antibody Identity card

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

HLy9.1.25

  • Clone name HLy9.1.25
  • Description Mouse monoclonal
  • Antigen used 300.19 cell line transfected with the CD229 full-length DNA
  • Epitope Unknown
  • Isotype IgG1, κ
  • Confirmed species reactivity Human
  • Ab used Biolegend (Cat. 326108) 50 μg/ml
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)1 μg/mlNot workingOdyssey Western Blotting protocol (OdWB)
Immunocytochemistry (ICC)1.6 μg/mlWorkingICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)1 μg/mlNot workingBOND-MAX automated immunohistochemistry
Immunoflourescence (IF)1 μg/mlWorkingImmunofluorescence staining
Flow cytometry (FC)0.5 μg/mlWorkingFlow cytometry
IHC-P Species1.6 μg/mlNot workingBOND-MAX automated immunohistochemistry

Ab ID Card application validation / characterisation

WB Validation
Over-expression/cross reactivity
Not working
Gene inactivation
Not working
WB Characterisation
Endogenous expression
Not working
ICC / ICC-P Validation
Over-expression/cross reactivity
ICC Validation: Over-expression/cross reactivity
Immunocytochemistry (ICC) of the HLy9.1.25 mAb was performed on frozen cytospin preparations of GFP-tagged human CD229/LY9 and MYC-tagged LILRB3 proteins expressed in transfected HEK293T cells. Labelling with the HLy9.1.25 mAb was present in the HEK-hLY9-GFP transfectants but not in the negative control HEK-LILRB3-MYC cells. Labelling with anti-MYC and anti-GFP were used to confirm the efficiency of transfection.

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Gene inactivation
ICC Validation: Gene inactivation
The specificity of the HLy9.1.25 mAb for the endogenous CD229/Ly9 protein was confirmed by ICC on cytospin preparations of the U266 cell line before (U266 WT) and after LY9 gene inactivation (U266 KO LY9) using CRISPR-Cas9 technology. CD229/Ly9 membrane expression was observed in U266 WT while no staining was observed in U266 KO LY9 cells.

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IHC-P Characterisation
Endogenous expression
Not working
IF Characterisation
Endogenous expression
IF Characterisation: Endogenous expression
Double immunofluorescence for CD229/LY9 using the HLy9.1.25 mAB (red) and either anti-CD3 or anti-CD45 (green) on Jurkat-Raji cell conjugates which had been incubated for 10 min in the presence of SEE. Phase contrast images merged with blue fluorescence from the Blue-CMAC-loaded Jurkat-Raji cell conjugates are shown. Note the double labelling at points of cell contact.

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FC Validation
Over-expression/cross reactivity
FC Validation: Over-expression/cross reactivity
Overexpression of CD229/LY9 protein was demonstrated using the HLy9.1.25 mAb by flow cytometry. HEK293T cells transfected with hCD229/LY9 were used as a positive control (light blue) while HEK293T cell transfected with CLEC5a were used as a negative control (pink).

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Gene inactivation
FC Validation: Gene inactivation
The specificity of the HLy9.25 mAb for the endogenous CD229/LY9 protein was confirmed by flow cytometry using cell extracts of U266 cell line before (U266 wt, light blue) and after hLY9 gene inactivation (U266 ko ly9, pink) using CRISPR-Cas9 technology.

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FC Characterisation
Endogenous expression
FC Characterisation: Endogenous expression
Flow cytometry of the U266, L363, ARP-1, SU-DHL-1 and Karpas 299 lymphoma-derived cells line using the HLy9.1.25 mAb to detect CD229/LY9. Positive labelling is seen in the U266, L363 and ARP-1 cell lines.

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IHC-P Domestic species
Not working
IHC-P Wild species
Not working
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