Recombinant GST and His-tagged Protein Purification

GST, GST-PRMT5, GST-PRMT5 truncated recombinant proteins were expressed in Escherichia coli BL21-DE3 by induction with 1 mM IPTG (Amersco, SF, USA). GST-tagged proteins were purified using Glutathione Sepharose 4B beads (GE Healthcare). G3BP2-His and G3BP2-His truncated proteins were purified with Ni-NTA agarose beads (Qiagen). Different purified GST- and His-tagged recombinant proteins were incubated with pull-down buffer (50 mM Tris-Cl, pH 8.0, 200 mM NaCl, 10 mM MgCl₂, 1% NP-40, 1 mM EDTA,1 mM DTT) for 2 h at 4 °C before immunoblotting assay.

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Wang N., Li T., Liu W., Lin J., Zhang K., Li Z., Huang Y., Shi Y., Xu M, & Liu X. (2023). USP7- and PRMT5-dependent G3BP2 stabilization drives de novo lipogenesis and tumorigenesis of HNSC. Cell Death & Disease, 14(3), 182.

Publication 2023

Glutathione Sepharose 4B beads Ni-NTA agarose beads

Agarose Buffer Edta Escherichia coli Glutathione Immunoblotting assay Iptg Mgcl2 Nacl Np 40 Proteins Pull Recombinant proteins Sepharose 4b Tris

Corresponding Organization : Jiaying University

Other organizations : Sun Yat-sen University, Sun Yat-sen University Cancer Center

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Variable analysis

independent variables

Expression of GST, GST-PRMT5, GST-PRMT5 truncated recombinant proteins in Escherichia coli BL21-DE3 by induction with 1 mM IPTG
Purification of GST-tagged proteins using Glutathione Sepharose 4B beads
Purification of G3BP2-His and G3BP2-His truncated proteins using Ni-NTA agarose beads
Incubation of purified GST- and His-tagged recombinant proteins with pull-down buffer

dependent variables

Immunoblotting assay

control variables

Pull-down buffer composition (50 mM Tris-Cl, pH 8.0, 200 mM NaCl, 10 mM MgCl2, 1% NP-40, 1 mM EDTA, 1 mM DTT)
Incubation time (2 h) and temperature (4 °C)

controls

No positive or negative controls were explicitly mentioned in the provided information.

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