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EuroMAbNet: European Monoclonal Antibodies Network

Antibody Identity card

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

UYSY20

Clone name UYSY20
Description Rat monoclonal
Antigen used Unknown
Epitope Unknown
Isotype IgG2a
Confirmed species reactivity Human and mouse
Ab used Creative Diagnostics (Cat. CABT-BL8406) 0.5 mg/ml

APPLICATION	Recommended concentration	Status	Protocol
Western blotting (WB)	2.5 ug/ml	Working	Odyssey Western Blotting protocol (OdWB)
Immunocytochemistry (ICC)	0.75 ug/ml	Not working	ICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)	5 μg/ml	Working	BOND-MAX automated immunohistochemistry
Immunoflourescence (IF)	12.5 ug/ml	Working	Immunofluorescence staining
Flow cytometry (FC)	5 ug/ml	Working	Flow cytometry
IHC-P Species	6.25 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 (WB) using the UYSY20 mAb was performed on cell extracts of MYC-FLAG tagged human TOX, MYC-tagged TOX2 and GFP-tagged TOX3 and TOX4 transfected HEK293T cells. Two bands of 68 kDa and 57 kDa were detected in the HEK-TOX cell extracts while no expression was found in the HEK-TOX2, HEK-TOX3 and HEK-TOX4 extracts. Anti-GAPDH was used as loading control.

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Gene inactivation

WB Validation: Gene inactivation — The specificity of the UYSY20 mAb for endogenous TOX protein was confirmed by WB using cell extracts of the MOLT4 cell line before and after TOX gene inactivation (MOLT4 KO TOX) using CRISPR-Cas9 technology. Anti-GAPDH was used as loading control.

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WB Characterisation

Endogenous expression

WB Characterisation: Endogenous expression — WB using the UYSY20 mAb to confirm endogenous expression of TOX showed bands of 63 kDa and 57 kDa of TOX protein in the SUDHL4 diffuse large B-cell lymphoma cells. TOX was not expressed in the U266 myeloma cell line. Anti-GAPDH was used as loading control.

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ICC / ICC-P Validation

Over-expression/cross reactivity

ICC Validation: Over-expression/cross reactivity — To confirm the lack of cross-reactivity with the other TOX family members, immunohistochemistry (ICC) with the UYSY20 mAb was performed on frozen cytospin preparations of MYC-FLAG tagged human TOX, MYC-tagged TOX2 and GFP-tagged TOX3 and TOX4 proteins expressed in HEK293T cells. Labelling with the anti-MYC and anti-GFP confirmed the efficiency of transfection.

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Gene inactivation

Not working

IHC-P Characterisation

Endogenous expression

IHC-P Characterisation: Endogenous expression — Single immunoperoxidase labeling in tonsil of the UYSY20 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 using the UYSY20 mAb (green) with 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 lack of cross-reactivity with the other TOX family members, flow cytometry with the UYSY20 mAb was performed on wild type HEK293T (red) and HEK293T cells transfected with one of the following: MYC-tagged human TOX (light blue), TOX2 (orange), 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 UYSY20 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 TO, blue) using CRISPR-Cas9 technology.

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FC Characterisation

Endogenous expression

FC Characterisation: Endogenous expression — Flow cytometry using the UYSY20 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 using the UYSY20 mAb on paraffin sections of lymph node from a variety of 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 — Immunoperoxidase staining using the UYSY20 mAb on paraffin sections of lymph node from a variety of wild exotic animal species.

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