GST Pulldown Assay for Protein Interactions

The GST pulldown assay was performed as previously described^{68 (link)}. GST or GST-ELF5 fusion protein was expressed in E. coli BL21 (Takara, Tokyo, JAPAN) and induced with 0.1 mM Isopropyl β-D-Thiogalactoside (IPTG) at 28 °C for 6 h. Then GST or GST-ELF5 fusion protein purified with Pierce GST Spin Purification Kit (Thermo Scientific, Massachusetts, USA). The purified proteins were immobilized on the Pierce Spin Column and then incubated with HEK293T cells lysate with overexpressing p300 or SIRT6.

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Li X., Li S., Li B., Li Y., Aman S., Xia K., Yang Y., Ahmad B, & Wu H. (2021). Acetylation of ELF5 suppresses breast cancer progression by promoting its degradation and targeting CCND1. NPJ Precision Oncology, 5, 20.

Publication 2021

E. coli BL21 Pierce GST Spin Purification Kit

Assay Cells E coli Isopropyl thiogalactoside P300 Proteins Sirt6

Corresponding Organization :

Other organizations : Dalian University of Technology

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Variable analysis

independent variables

Expression of GST or GST-ELF5 fusion protein
Overexpression of p300 or SIRT6 in HEK293T cell lysate

dependent variables

Binding of GST or GST-ELF5 fusion protein to overexpressed p300 or SIRT6 in HEK293T cell lysate

control variables

Induction of GST or GST-ELF5 fusion protein expression in E. coli BL21 using 0.1 mM IPTG at 28°C for 6 hours
Purification of GST or GST-ELF5 fusion protein using Pierce GST Spin Purification Kit
Immobilization of purified proteins on Pierce Spin Column

positive controls

Incubation of immobilized GST-ELF5 fusion protein with HEK293T cell lysate overexpressing p300 or SIRT6

negative controls

Incubation of immobilized GST protein with HEK293T cell lysate overexpressing p300 or SIRT6

Annotations

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