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Antibody Identity card

Antibody information [IL4I1 ]

BALI573B

  • Clone name BALI573B
  • Description Rat monoclonal
  • Antigen used Human IL4I1-His-Strep expressed in 293 cells
  • Epitope Unknown
  • Isotype IgG2a
  • Confirmed species reactivity Human
  • Ab used Tissue culture supernatant
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)WorkingWestern Blotting (WB)
Immunocytochemistry (ICC)Neat supernatantWorkingICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)1:10 SupernatantWorkingBOND-MAX automated immunohistochemistry
Immunoflourescence (IF)1:2 culture supernatantWorkingImmunofluorescence protocol for culture cells
Flow cytometry (FC)2:5 supernatantNot workingFlow cytometry
IHC-P SpeciesNot tested

Ab ID Card application validation / characterisation

WB Validation
Over-expression/cross reactivity
IL4I1 antibody WB Validation: Over-expression/cross reactivity
To confirm the specificity of the antibody, Western blotting (WB) of the BALI573B mAb was performed using cell extracts of transfected HEK cells. A 70 kDa was detected only in the HEK-hIL4I1 cell extracts expressing human IL4I1 and not in HEK cells expressing murine IL4I1 (HEK-mIL4I1-His) or human LY9 (HEK-hLY9). Anti-GAPDH was used as loading control.

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Gene inactivation
IL4I1 antibody WB Validation: Gene inactivation
WB of BALI573B mAb using cell extracts of U266 before (U266) and after gene inactivation (U266 KO IL4I1) using Crispr-Cas9 technology. Anti vinculin was used as loading control.

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WB Characterisation
Endogenous expression
IL4I1 antibody WB Characterisation: Endogenous expression
WB using the BALI573B mAb showed 70 kDa bands of IL4I1 protein in extracts of the U266 (myeloma) and DEV (Hodgkin lymphoma) cell lines. No bands were observed in total tonsil extract or the RAW (mouse macrophage) cell line. Anti-GAPDH was used as a loading control.

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ICC / ICC-P Validation
Over-expression/cross reactivity
IL4I1 antibody ICC Validation: Over-expression/cross reactivity
To confirm lack of cross-reactivity with the mouse Il4I1, immunocytochemistry (ICC) with the BALI573B mAb was performed on frozen cytospin preparations of HIS-tagged human IL4I1 and mouse IL4I1 expressed in HEK293T cells. Staining was observed only of the HEK-hIL4I1-HIS cells showing that BALI573B is specific for human IlL4I1. Labelling with the anti-HIS confirmed the efficiency of transfection

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Gene inactivation
IL4I1 antibody ICC Validation: Gene inactivation
The specificity of the BALI573B mAb for endogenous IL4I1 protein was confirmed by immunohistochemistry (IHC-P) on sections of paraffin-fixed U266 cells before and after IL4I1 gene inactivation using CRISPR-Cas9 technology. Cytoplasmic expression was observed in the the wild type U266 cell line, while no staining was observed in gene inactivated U266 cells (U266 KO IL4I1).

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IHC-P Characterisation
Endogenous expression
IL4I1 antibody IHC-P Characterisation: Endogenous expression
Single immunoperoxidase labelling with the BALI573B mAb showed cytoplasmic and membrane staining of IL4I1 protein in germinal centre macrophages and mature dendritic cells within the interfollicular area of the tonsil. The IL4I1 protein was also observed (weaker staining) in B cells of the germinal centre, particularly in the dark zone. IL4I1 was found in macrophages in the spleen and Burkitt lymphoma. Additionally, strong staining was found in striated muscle and the surface of epithelium in the small intestine.

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IF Characterisation
Endogenous expression
IL4I1 antibody IF Characterisation: Endogenous expression
Double immunofluorescence staining of human paraffin embedded tonsil by the BALI573B mAb (red) and anti-CD68 (green). Cell nuclei are marked with DAPI (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 tested
IHC-P Wild species
Not tested
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