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EuroMAbNet: European Monoclonal Antibodies Network

Antibody Identity card

Antibody information [TOX (Thymocyte Selection Associated High Mobility Group Box)]

NAN448B

Clone name NAN448B
Description Rat monoclonal
Antigen used His-TOX (aa 1-250) recombinant protein
Epitope LLPFHPQNMDL
Isotype IgG1
Confirmed species reactivity Human
Ab used TOX clone NAN448 CNIO (purified mAb 3 mg/ml)

APPLICATION	Recommended concentration	Status	Protocol
Western blotting (WB)	4 ug/ml	Working	Odyssey Western Blotting protocol (OdWB)
Immunocytochemistry (ICC)	2.5 ug/ml	Working	ICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)	4 ug/ml	Working	BOND-MAX automated immunohistochemistry
Immunoflourescence (IF)	7.5 ug/ml	Working	Immunofluorescence protocol for culture cells
Flow cytometry (FC)	5 ug/ml	Working	Flow cytometry
IHC-P Species	3 ug/ml	Working	BOND-MAX automated immunohistochemistry

Ab ID Card application validation / characterisation

WB Validation

Over-expression/cross reactivity

WB Validation: Over-expression/cross reactivity — To confirm the lack of cross-reactivity with the other TOX family members, Western blotting of the NAN448B mAb was performed using cell extracts of MYC-FLAG tagged human TOX, MYC-tagged TOX2 and GFP-tagged TOX3 and TOX4. Two bands of 68 kDa and 57 kDa were detected by WB only in the HEK-TOX cell extracts. Anti-GAPDH was used as loading control.

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Gene inactivation

WB Validation: Gene inactivation — The specificity of the NAN448B mAb for the endogenous TOX protein was confirmed by WB using cell extracts of MOLT4 cell line before (MOLT4 WT) and after TOX gene inactivation (MOLT4 KO TOX) 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 NAN448B mAb showed 68 kDa and 57 kDa bands of TOX protein in the SUDHL4 diffuse large B-cell lymphoma cell line. TOX was not expressed in the U266 myeloma cell line. Anti-GAPDH was used as a loading control.

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ICC / ICC-P Validation

Over-expression/cross reactivity

ICC Validation: Over-expression/cross reactivity — To confirm lack of cross-reactivity with the other TOX family members, immunocytochemistry (ICC) with the NAN448B mAb was performed on frozen cytospin preparations of MYC-tagged human TOX and TOX2 and GFP-tagged TOX3 and TOX4 proteins expressed in HEK293T cells. Staining was observed only of the TOX transfectants. Labelling with the anti-MYC and anti-GFP confirmed the efficiency of transfection.

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Gene inactivation

ICC Validation: Gene inactivation — The specificity of the NAN448B mAb for the endogenous TOX protein was confirmed by ICC in cytospin preparations of the MOLT4 cell line before and after (MOLT4 KO TOX) gene inactivation using CRISPR-Cas9 technology. TOX nuclear expression was observed in wild type MOLT4 cells, while no staining was observed in the MOLT4 KO TOX cells.

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IHC-P Characterisation

Endogenous expression

IHC-P Characterisation: Endogenous expression — Single immunoperoxidase labelling in tonsil of the NAN448B mAb shows strong nuclear staining of TOX in germinal centre cells and cells in the interfollicular area. (B) TOX is also highly expressed by cells in the thymic cortex. (C) In spleen, TOX is expressed by scattered cells in the white and red pulp.

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IF Characterisation

Endogenous expression

IF Characterisation: Endogenous expression — Triple immunofluorescence staining of human paraffin embedded tonsil by the NAN448B mAb (green), anti-CD20 (red) and anti-CD3 (white).

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FC Validation

Over-expression/cross reactivity

FC Validation: Over-expression/cross reactivity — To confirm the lack of cross-reactivity with the other TOX family members, flow cytometry with the NAN448B mAb was performed on wild type HEK293T (red) and HEK293T cells transfected with MYC-tagged human TOX (light blue) and TOX2 (orange) and GFP-tagged TOX3 (light green) and TOX4 (dark green) plasmid vectors.

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Gene inactivation

FC Validation: Gene inactivation — The specificity of the NAN448B mAb for detecting endogenous TOX protein was confirmed by flow cytometry using a MOLT4 cell line before (red) and after TOX gene inactivation (MOLT4 KO TOX, blue) using CRISPR-Cas9 technology.

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FC Characterisation

Endogenous expression

FC Characterisation: Endogenous expression — Flow cytometry using the NAN448B mAb shows endogenous TOX protein in the MOLT4 cells (red) but not in the U266 cells (blue).

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IHC-P Domestic species

Canary Serinus canaria: +
Cat Felis catus: -
Cow Bos taurus: +++
Dog Canis lupus familiaris: +++
Goat Capra aegagrus: +++
Horse Equus ferus: +++
Jacobin Pigeon Columba livia: ++
Llama Llama glama: +++
Mouse Mus musculus: +++
Pig Sus scrofa domesticus: +++
Rat Rattus norvegicus: -
Sheep Ovis orientalis: +++

IHC-P Domestic species — Immunoperoxidase staining of the NAN448B mAb in paraffin sections of lymph node from domestic animal species.

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IHC-P Wild species

African sacred ibis Threskiornis aethiopicus: +++
Asian black bear Ursus thibetanus: +++
Baboon Papio sp.: +++
Badger Meles meles: +++
Bush Dog Speothos venaticus: +
Common brown lemur Eulemur fulvus mayottensis: +++
Emu Dromaius novaehollandiae: +
European bison Bison bonasus: ++
Mandrill Mandrillus sphinx: +++
Otter Pteronura brasiliensis: -
Patagonian mara Dolichotis patagonum: +++
Penguin Spheniscus demersus: +
Raccoon Procyon lotor: +++
Red flamimgo Phoenicopterus ruber: ++
Reindeer Rangifer tarandus: +++
Rhea Rhea americana: +
Sandbar shark Carcharhinus plumbeus: -
Sitatunga Tragelaphus spekii: +
South American sea lion Otaria byronia: ++
Stork Ciconia ciconia: -
Wild pig Sus scrofa: ++
Wolf Canis lupus: +

IHC-P Wild species — Immunohistochemistry staining of NAN448B mAb in paraffin section of lymph node of exotic wild animal species.

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