Purification and Pulldown of GST-Fusion Proteins

GST alone, GST-KLF12 and GST-p53 were expressed in Escherichia coli BL21 (Takara, Dalian, China) and purified using Pierce GST Spin Purification Kit (Thermo Scientific, MA, USA) as per the manufacturer’s instruction. The purified GST and GST-KLF12 were seperately immobilized on a different Pierce Spin Column and used to pull down endogenous p53 from MCF-7 cell lysate. The purified GST and GST-p53 were seperately immobilized on a different Pierce Spin Column and used to pull down exogenous p300 from HEK293T cell lysate. An immunofluorescence assay was conducted as described previously [63 (link)].

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Li Y., Li S., Shi X., Xin Z., Yang Y., Zhao B., Li Y., Lv L., Ren P, & Wu H. (2023). KLF12 promotes the proliferation of breast cancer cells by reducing the transcription of p21 in a p53-dependent and p53-independent manner. Cell Death & Disease, 14(5), 313.

Publication 2023

Escherichia coli BL21 Pierce GST Spin Purification Kit

Cell Escherichia coli Immunofluorescence assay Mcf 7 cell P300

Corresponding Organization :

Other organizations : Dalian University of Technology, Second Affiliated Hospital of Dalian Medical University, Dalian Medical University

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Variable analysis

independent variables

Expression of GST alone, GST-KLF12, and GST-p53 in Escherichia coli BL21
Immobilization of purified GST and GST-KLF12 on different Pierce Spin Columns
Immobilization of purified GST and GST-p53 on different Pierce Spin Columns

dependent variables

Endogenous p53 pulled down from MCF-7 cell lysate
Exogenous p300 pulled down from HEK293T cell lysate
Results of the immunofluorescence assay

control variables

Escherichia coli BL21 cell line used for protein expression
Pierce GST Spin Purification Kit used for protein purification
Pierce Spin Columns used for protein immobilization
MCF-7 and HEK293T cell lines used for cell lysate preparation

controls

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

Annotations

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