Purification of N-terminal GST fusion proteins with immobilized glutathione

Genes cloned into pT7CFE1-NHis-GST-CHA were used to express GST-fusion proteins for 6 hours. After binding to glutathione agarose, the GST-fused proteins were eluted with 50 mM glutathione. The additional bands (denoted with arrows) found with the purified proteins were previously identified as cellular proteins eEF1G and GSTM3 by mass spectrometry which are known to bind to the glutathione column and co-elute with GST tagged proteins. Approximately 500 ng of each of the purified proteins was separated by SDS-PAGE and stained with the Pierce Power Stainer (Cat# 22833). Abbreviations: His: repeat of 6X Histidines; GST: Glutathione S Transferase; HRV3c site: Human Rhinovirus 3C Protease site; GOI: gene of interest; HA: Haemaglutunnin sequence; BSA: Bovine serum albumin; GFP: Green fluorescent protein; CAV1: caveolin1; BAD: Bcl-2-associated death promoter; Green Renilla: Green Renilla luciferase; CCND1: cyclin D1; Guassia: Guassia luciferase; AKT3: protein kinase; AGO2: Argonaute RISC catalytic component 2; and RB1: Retinoblastoma 1.

Genes cloned into pT7CFE1-NHis-GST-CHA were used to express GST-fusion proteins for 6 hours. After binding to glutathione agarose, the GST-fused proteins were eluted with 50 mM glutathione. The additional bands (denoted with arrows) found with the purified proteins were previously identified as cellular proteins eEF1G and GSTM3 by mass spectrometry which are known to bind to the glutathione column and co-elute with GST tagged proteins. Approximately 500 ng of each of the purified proteins was separated by SDS-PAGE and stained with the Pierce Power Stainer (Cat# 22833). Abbreviations: His: repeat of 6X Histidines; GST: Glutathione S Transferase; HRV3c site: Human Rhinovirus 3C Protease site; GOI: gene of interest; HA: Haemaglutunnin sequence; BSA: Bovine serum albumin; GFP: Green fluorescent protein; CAV1: caveolin1; BAD: Bcl-2-associated death promoter; Green Renilla: Green Renilla luciferase; CCND1: cyclin D1; Guassia: Guassia luciferase; AKT3: protein kinase; AGO2: Argonaute RISC catalytic component 2; and RB1: Retinoblastoma 1.

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