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Antibody Identity card

Antibody information [CD305 Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1)]

JAVI82A

  • Clone name JAVI82A
  • Description Rat monoclonal
  • Antigen used RBL-1-LAIR1-MYC-DDK transfected cells and last booster with LAIR1 recombinant (Gln22-His163, with a C-terminal 6-His tag)
  • Epitope Unknown
  • Isotype IgG2a, K
  • Confirmed species reactivity Human
  • Ab used CNIO
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)No dilution (neat supernatant)WorkingWestern Blotting (WB)
Immunocytochemistry (ICC)No dilution (neat supernatant)WorkingICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)1:5 culture supernatantWorkingBOND-MAX automated immunohistochemistry
Immunoflourescence (IF)1:2 culture supernatantWorkingImmunofluorescence staining
Flow cytometry (FC)20ul of culture supernatant/tubeWorkingFlow cytometry
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) using the JAVI82A mAb and cell extracts of HEK293T cells overexpressing human LAIR1 (HEK-LAIR1-MYC) or LAIR2 (HEK-LAIR2-MYC) proteins was used to confirm the specificity of the antibody for LAIR1. Two bands of about 40 and 48 kDa was found only in the LAIR1 transfected cells. Anti-vinculin was used as the loading control.

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Gene inactivation
WB Validation: Gene inactivation
The specificity of the JAVI82A mAb for the endogenous LAIR1 protein was confirmed by WB using cell extracts of THP1cell line before (THP1) and after LAIR1 gene inactivation (LAIR1 KO THP1) using CRISPR-Cas9 technology. Anti-vinculin was used as loading control.

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WB Characterisation
Endogenous expression
WB Characterisation: Endogenous expression
WB using the JAVI82A mAb showed 40 kDa bands of LAIR1 protein in lysates of U937 (myeloid) and MOLT4 (T-cell leukaemia) cells and tonsil but absent in JURKAT (T-lymphoblastic) cells. 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 JAVI82A mAb was performed on frozen cytospin preparations of MYC-tagged LAIR1 and MYC-tagged LAIR2 proteins expressed in HEK293T cells. Staining was detected only in the HEK-LAIR1-MYC transfectants. Labelling with anti-MYC was used to confirm the efficiency of transfection.

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Gene inactivation
ICC Validation: Gene inactivation
The specificity of the JAVI82A mAb for the endogenous LAIR1 protein was confirmed by ICC on sections of formalin fixed paraffin embedded pellets of the THP1 cell line before (THP1 WT) and after (LAIR1 KO THP1) gene inactivation using CRISPR-Cas9 technology. LAIR1 membrane expression was observed in wild type THP1 cells, while no staining was observed in the LAIR1 KO THP1 cells.

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IHC-P Characterisation
Endogenous expression
IHC-P Characterisation: Endogenous expression
Single immunoperoxidase labelling with the JAVI82A mAb shows cytoplasmic and membrane staining of the LAIR1 protein in human tonsil, specifically in the B- and T- cell areas, except for germinal centre B cells. In the spleen, LAIR1 is expressed in both B- and T- cells. In the thymus, strong staining is observed in the cortex, with the medulla also showing positivity. Staining for LAIR1 is observed in anaplastic large cell lymphoma (ALCL).

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IF Characterisation
Endogenous expression
IF Characterisation: Endogenous expression
Double immunofluorescence labelling of paraffin fixed human tonsil with the JAVI82A mAb (red), anti-CD68 (green) and DAPI (blue).

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FC Validation
Over-expression/cross reactivity
FC Validation: Over-expression/cross reactivity
Overexpression of LAIR1 protein was demonstrated using mAb JAVI82A in flow cytometry. HEK293T cells transfected with LAIR1 were used as a positive control (light blue) while HEK293T cell transfected with LAIR2 were used as a negative control (pink). Control experiments using only the secondary antibody show no staining for HEK LAIR2 cells (discontinuous pink line) and HEK LAIR11 cells (discontinuous blue line).

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Gene inactivation
FC Validation: Gene inactivation
The specificity of the JAVI82A mAb for the endogenous LAIR1 protein was confirmed by flow cytometry using THP1 cell line before (THP1_LAIR1, light blue) and after LAIR1 gene inactivation ( THP1 LAIR1 KO, pink) using CRISPR-Cas9 technology. Control experiments using only the secondary antibody show no staining for THP1 LAIR1 KO cells (discontinuous pink line) and THP1 cells (discontinuous blue line).

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FC Characterisation
Endogenous expression
FC Characterisation: Endogenous expression
Flow cytometry using the JAVI82A mAb shows endogenous LAIR1 protein in the THP1 cells (light blue) but not in the Raji cells (pink). Control experiments using only the secondary antibody show no staining for RAJI cells (discontinuous pink line) and THP1 cells (discontinuous blue line).

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IHC-P Domestic species
Not tested
IHC-P Wild species
Not tested
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