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Antibody Identity card

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

EPR24745-12A

  • Clone name EPR24745-12A
  • Description Recombinant rabbit monoclonal
  • Antigen used The exact immunogen used to generate this antibody is proprietary information
  • Epitope Unknown
  • Isotype IgG
  • Confirmed species reactivity Human
  • Ab used Abcam (Cat. ab315967) 1mg/ml
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)20 ug/ulWorkingWestern Blotting (WB)
Immunocytochemistry (ICC)10ug/ulWorkingICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)10 ug/ulWorkingBOND-MAX automated immunohistochemistry
Immunoflourescence (IF)20 ug/mlNot tested
Flow cytometry (FC)0.04ug/ulWorkingFlow 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) of the EPR24745-12A mAb was performed using cell extracts of transfected HEK293T cells overexpressing human LAIR1 (HEK-LAIR1-MYC) or human LAIR2 (HEK-LAIR2-MYC) proteins to confirm its specificity for LAIR1. Two bands of about 40 and 48 kDa were 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 EPR24745-12A mAb for the endogenous LAIR1 protein was confirmed by WB using cell extracts of the THP1 cell 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 EPR24745-12A mAb showed 40 kDa bands of LAIR1 protein in lysates of U937 (myeloid leukaemia) and MOLT4 (T-cell leukaemia) cell lines and tonsil. LAIR1 was not expressed in the JURKAT (T-cell leukaemia) cell line. 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 EPR24745-12A mAb was performed on frozen cytospin preparations of MYC-tagged LAIR1 and MYC-tagged LAIR2 proteins expressed in transfected HEK293T cells. Staining was detected only in the LAIR1 (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 EPR24745-12A mAb for endogenous LAIR1 protein was confirmed by ICC on sections of formalin fixed paraffin embedded wild type THP1 cells before (THP1) and after LAIR1 inactivation (LAIR1_KO THP1) using CRISPR-Cas9 technology. Labelling was only observed in the wild type THP1 cells.

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IHC-P Characterisation
Endogenous expression
IHC-P Characterisation: Endogenous expression
Single immunoperoxidase labelling with the EPR24745-12A mAb shows cytoplasmic and membrane staining of the LAIR1 protein in human tonsil and lymph node, specifically in the B- and T- cell areas, except for germinal centre B cells. In the spleen, LAIR1 is expressed in B- and T-cell areas with the exception of germinal centre cells.

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IF Characterisation
Endogenous expression
IF Characterisation: Endogenous expression
Double immunofluorescence labelling of paraffin fixed human tonsil with the EPR24745-12A 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 EPR24745-12A 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 LAIR1 cells (discontinuous blue line).

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Gene inactivation
FC Validation: Gene inactivation
The specificity of the EPR24745-12A mAb for the endogenous LAIR1 protein was confirmed by flow cytometry using THP1 cell line before (THP1, light blue) and after LAIR1 gene inactivation (LAIR1 KO THP1, pink) using CRISPR-Cas9 technology. Control experiments using only the secondary antibody show no staining for LAIR1 KO THP1 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 EPR24745-12A 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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