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Antibody Identity card

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

H-2

  • Clone name H-2
  • Description Mouse monoclonal
  • Antigen used 31-63 a.a near the N-terminus of human protein
  • Epitope Unknown
  • Isotype IgG3
  • Confirmed species reactivity Human
  • Ab used Santa Cruz sc-374137 (purified mAb 0.2 mg/ml)
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)6 ug/mlWorkingOdyssey Western Blotting protocol (OdWB)
Immunocytochemistry (ICC)2.5 ug/mlWorkingICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)2.5 ug/mlNot workingBOND-MAX automated immunohistochemistry
Immunoflourescence (IF)2.5 ug/mlNot workingImmunofluorescence staining
Flow cytometry (FC)2.5 ug/mlNot workingFlow cytometry
IHC-P Species2.5 ug/mlNot workingBOND-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 of the H-2 mAb with the other TOX family members, western blotting (WB) 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 in HEK-TOX cell extracts while no staining was found in HEK-TOX2, HEK-TOX3 and HEK-TOX4. Anti-GAPDH was used as loading control.

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Gene inactivation
WB Validation: Gene inactivation
The specificity of the H-2 mAb for the endogenous TOX protein was confirmed by WB using cell extracts of MOLT4 cell line before 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 H-2 mAb showed a 68 kDa and a weaker 57 kDa band in the SUDHL4 diffuse large B-cell line. 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, immunocytochemistry (ICC) with the H-2 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 anti-MYC and anti-GFP confirmed the efficiency of transfection.

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Gene inactivation
Not tested
IHC-P Characterisation
Endogenous expression
Not working
IF Characterisation
Endogenous expression
Not working
FC Validation
Over-expression/cross reactivity
Not working
Gene inactivation
Not working
FC Characterisation
Endogenous expression
Not working
IHC-P Domestic species
Not working
IHC-P Wild species
Not working
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