Western Blot Analysis of Redox Proteins

Equal amounts of cell lysates were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Then, membranes were stained with indicated primary antibodies, as follows: mouse anti-human TRX1 (1:500, BD Biosciences, 559969), rabbit anti-human GLRX1 (1:1000, R&D Systems, AF3399), rabbit anti-human actin (1:1000, Sigma, A2066), or mouse anti-human GAPDH (1:2000, Ambion, 4300), followed by staining with IRDye 680 or IRDye 800-labeled secondary antibodies (1:10,000, LI-COR Biosciences). Membranes were then scanned using a LI-COR Odyssey scanner (LI-COR Biosciences) (18 (link)).

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Balta E., Janzen N., Kirchgessner H., Toufaki V., Orlik C., Liang J., Lairikyengbam D., Abken H., Niesler B., Müller-Decker K., Ruppert T, & Samstag Y. (2022). Expression of TRX1 optimizes the antitumor functions of human CAR T cells and confers resistance to a pro-oxidative tumor microenvironment. Frontiers in Immunology, 13, 1063313.

Publication 2022

IRDye 680 IRDye 800-labeled secondary antibodies LI-COR Odyssey scanner

Actin Antibodies Cell Gapdh Human Irdye 800 Membranes Mouse Polyacrylamide gels Polyvinylidene fluoride Rabbit Sodium dodecyl sulfate Trx1

Corresponding Organization :

Other organizations : University Hospital Heidelberg, German Cancer Research Center, European Molecular Biology Laboratory

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Levels of TRX1 protein
Levels of GLRX1 protein
Levels of actin protein
Levels of GAPDH protein

control variables

Equal amounts of cell lysates
SDS-polyacrylamide gel electrophoresis
Transfer to polyvinylidene fluoride membranes
Primary antibodies: mouse anti-human TRX1 (1:500, BD Biosciences, 559969), rabbit anti-human GLRX1 (1:1000, R&D Systems, AF3399), rabbit anti-human actin (1:1000, Sigma, A2066), or mouse anti-human GAPDH (1:2000, Ambion, 4300)
Secondary antibodies: IRDye 680 or IRDye 800-labeled (1:10,000, LI-COR Biosciences)
Scanning with LI-COR Odyssey scanner (LI-COR Biosciences)

controls

No positive or negative controls were explicitly mentioned.

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