Purification and Binding Assay of GST-tagged Proteins

GST, GST-MUC1-CD, and GST-MUC1-CD(AQA) were prepared as described (28 (link)). GST-tagged BMI1 was generated by PCR amplification of the pT3-EF1a-BMI1 plasmid (Addgene) and subcloning into the pGEX-5X-1 expression vector (GE Healthcare, Pittsburg, PA, USA). Purified GST-MUC1-CD and GST-MUC1-CD(AQA) were cleaved with thrombin to remove the GST moiety (30 (link)). For bindings assays, purified proteins were incubated for 2 h at room temperature. Adsorbates to glutathione-conjugated beads were analyzed by immunoblotting.

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Hiraki M., Maeda T., Bouillez A., Alam M., Tagde A., Hinohara K., Suzuki Y., Markert T., Miyo M., Komura K., Ahmad R., Rajabi H, & Kufe D. (2016). MUC1-C ACTIVATES BMI1 IN HUMAN CANCER CELLS. Oncogene, 36(20), 2791-2801.

Publication 2016

pT3-EF1a-BMI1 plasmid pGEX-5X-1 expression vector

Assays Bmi1 Glutathione Muc1 Plasmid Proteins Thrombin Vector

Corresponding Organization :

Other organizations : Dana-Farber Cancer Institute, Harvard University

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Variable analysis

independent variables

Preparation of GST, GST-MUC1-CD, and GST-MUC1-CD(AQA) as described (28)
Generation of GST-tagged BMI1 by PCR amplification and subcloning into the pGEX-5X-1 expression vector

dependent variables

Binding of purified proteins in binding assays

control variables

Incubation of purified proteins for 2 h at room temperature
Analysis of adsorbates to glutathione-conjugated beads by immunoblotting

controls

None specified
None specified

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