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Antibody Identity card

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

6E6D03

  • Clone name 6E6D03
  • Description Mouse monoclonal
  • Antigen used Human TOX recombinant protein (66-301 a.a.)
  • Epitope aa 66-301
  • Isotype IgG1
  • Confirmed species reactivity Human
  • Ab used Biolegend Cat.682602 (0.5 mg/ml)
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)2.5 μg/mlWorkingOdyssey Western Blotting protocol (OdWB)
Immunocytochemistry (ICC)2.5 μg/mlWorkingICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)2.5 μg/mlNot workingBOND-MAX automated immunohistochemistry
Immunoflourescence (IF)2.5 μg/mlNot workingImmunofluorescence staining
Flow cytometry (FC)5 μg/mlNot workingFlow cytometry
IHC-P Species2.5 μg/mlNot workingBOND-MAX automated immunohistochemistry

Ab ID Card application validation / characterisation

WB Validation
Over-expression/cross reactivity
WB Validation: Over-expression/cross reactivity
Western blotting (WB) was performed with the 6E6D03 mAb to confirm its lack of cross-reactivity using cell extracts of transfected HEK293T cells overexpressing MYC-FLAG tagged human TOX, MYC-tagged TOX2 and GFP-tagged TOX3 and TOX4 proteins. Two bands of 68 kDa and 57 kDa were detected in HEK-TOX, 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 6E6D03 mAb for the 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
Western blotting using the 6E6D03 mAb showed the presence of a 68 kDa TOX protein band 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 the lack of cross-reactivity with the other TOX family members, immunocytochemistry (ICC) with the 6E6D03 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 in the transfectants expressing TOX protein. 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
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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