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EuroMAbNet: European Monoclonal Antibodies Network

Antibody Identity card

Antibody information [IL4I1 ]

1006231

Clone name 1006231
Description Rat monoclonal
Antigen used CHO derived hIL4i1 Met1-His567
Epitope Information not available
Isotype Rat IgG2a
Confirmed species reactivity Human
Ab used R&D Systems (cat MAB56843) 0.5 mg/ml

APPLICATION	Recommended concentration	Status	Protocol
Western blotting (WB)	2 ug/mL	Working	Western Blotting (WB)
Immunocytochemistry (ICC)	Neat tissue culture supernatant	Working	ICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)	0.41 ug/ml	Working
Immunoflourescence (IF)	1.25 ug/ml	Working	Immunofluorescence staining
Flow cytometry (FC)	2.5 ug/ml	Working	Flow cytometry
IHC-P Species		Not tested

Ab ID Card application validation / characterisation

WB Validation

Over-expression/cross reactivity

IL4I1 antibody WB Validation: Over-expression/cross reactivity — Western blotting (WB) of the 1006231 mAb performed using cell extracts of transfected HEK293T cells. The expected 70kDa band was observed in cells expressing human IL4I1 protein (HEK hIL4I1) but not in cells transfected to express the mouse IL4I1 protein (HEK mIL4I1), indicating its specificity for the human protein. No band was seen in transfected cells expressing an unrelated protein (HEK TOX). Anti-vinculin was used as a loading control.

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Gene inactivation

IL4I1 antibody WB Validation: Gene inactivation — WB of the 1006231 mAb using cell extracts of U266 before (U266-WT) and after gene inactivation (U266-KO) and activated and unactivated THP1 before (THP1-WT and THP1-WT-MO) and after IL4I1 gene inactivation (THP1-KO MO) using Crispr-Cas9 technology. THP1 activation to macrophages (THPI-WT MO) was via treatment with PMA, IL-4 and IL-13. As expected a specific 70kDa band is observed in the WT cell extracts of U266 and THP1-WT MO. Activation of THP1 WT to macrophages (MO) was necessary for IL4I1 expression in the THP1 cell line since no band was observed in the THP1-WT cell lysate. No bands are observed in the KO or untreated cell extracts. Anti-tubulin was used as a loading control. Image kindly donated by Dr. Samanta Zanetti.

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WB Characterisation

Endogenous expression

IL4I1 antibody WB Characterisation: Endogenous expression — WB blotting of mAb 1006231 show endogenous expression of IL4I1 protein in the wild type and activated THP1 (monocytic leukaemia) cell line lysates. THP1 cells were activated to become M0, M1 and M2 macrophages via treatment with PMA, IFN-gamma, IL4, IL13 and/or LPS. As expected, increased expression of a specific 70kDa band was observed in the activated THP1 (THP1 MO, THP1 M1 and THP1 M2) cell lysates as compared to the unactivated THP1 cells. Anti-tubulin was used as a loading control. Image kindly donated by Dr. Samanta Zanetti.

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ICC / ICC-P Validation

Over-expression/cross reactivity

IL4I1 antibody ICC Validation: Over-expression/cross reactivity — Immunocytochemistry to confirm the specificity of the 1006231 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 1006231 mAb is specific for human IL4I1. No staining was seen of HEK cells transfected with mouse RAP1. 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 1006231 mAb for endogenous IL4I1 protein was confirmed by immunohistochemistry (IHC-P) on sections of formalin fixed paraffin embedded pellets of U266 cells before and after IL4I1 gene inactivation using CRISPR-Cas9 technology. Cytoplasmic expression was observed in the 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 1006231 mAb showed membrane staining of IL4I1 protein in germinal centre macrophages and mature dendritic cells within the interfollicular area of the tonsil. Scattered IL4I1-positive cells were observed in the spleen. Staining was detected in the Hassall's corpuscle and in macrophages in the thymus.

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IF Characterisation

Endogenous expression

IL4I1 antibody IF Characterisation: Endogenous expression — Double immunofluorescence staining of human paraffin embedded tonsil with Clone # 1006231 (red) and anti-CD68 (green). Cell nuclei are labelled with DAPI (blue).

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FC Validation

Over-expression/cross reactivity

IL4I1 antibody FC Validation: Over-expression/cross reactivity — Overexpression of IL4I1 protein was demonstrated using the 1006231 mAb in flow cytometry. HEK293T cells transfected with IL4I1 were used as a positive control (light blue) while HEK293T cell transfected with TRIB2 were used as a negative control (pink). Control experiments using only the secondary antibody show no staining for HEK TRIB2 cells (dashed pink line) and HEK IL4I1 cells (dashed blue line).

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Gene inactivation

IL4I1 antibody FC Validation: Gene inactivation — The specificity of the 1006231 mAb for the endogenous IL4I1 protein was confirmed by flow cytometry using U266 cell line before (U266, light blue) and after IL4I1 gene inactivation (Il4I1 KO U266, pink) using CRISPR-Cas9 technology. Control experiments using only the secondary antibody show no staining for Il4I1 KO U266 cells (dashed pink line) and U266 cells (dashed blue line).

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FC Characterisation

Endogenous expression

IL4I1 antibody FC Characterisation: Endogenous expression — Flow cytometry using the 1006231 mAb shows endogenous IL4I1 protein in the U266 cells (light blue) but not in the JURKAT cells (pink).Control experiments using only the secondary antibody show no staining for JURKAT cells (dashed pink line) and U266 cells (dashed blue line).

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IHC-P Domestic species

Not tested

IHC-P Wild species

Not tested

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