Purification of GST-tagged BRAT1 and WDR1

Escherichia coli BL-21 cells transformed with the pGEX-4T clone were cultured in 200 mL Luria-Bertani (LB) broth and treated with 1 mM IPTG for 3 h. The cells were collected in bacterial solution and lysed by sonication in BugBuster Master Mix (Novagen, San Diego, CA, USA). The lysates were then centrifuged at 13,000 × g and 4°C for 10 min. GST-tagged BRAT1 and GST-tagged WDR1 proteins were purified by glutathione-Sepharose column chromatography (GE Healthcare Life Sciences) and dialyzed as previously described (32 (link), 36 (link), 38 (link)).

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Hu L., Liu J., Shimada H., Ito M., Sugimoto K., Hiwasa T., Zhou Q., Li J., Shen S, & Wang H. (2022). Serum Anti-BRAT1 is a Common Molecular Biomarker for Gastrointestinal Cancers and Atherosclerosis. Frontiers in Oncology, 12, 870086.

Publication 2022

glutathione-Sepharose column chromatography

Bacterial Cells Clone Escherichia coli Glutathione Iptg Proteins Sepharose chromatography

Corresponding Organization : Chiba University

Other organizations : Jinan University, Toho University, Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine

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Variable analysis

independent variables

Addition of 1 mM IPTG

dependent variables

Purification of GST-tagged BRAT1 and GST-tagged WDR1 proteins

control variables

Escherichia coli BL-21 cells
Luria-Bertani (LB) broth
Sonication in BugBuster Master Mix
Centrifugation at 13,000 × g and 4°C for 10 min
Glutathione-Sepharose column chromatography
Dialysis of purified proteins

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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