GST Pulldown技术检测HEK293T细胞活化的Rab35

目的:运用GST pulldown技术建立真核细胞中活化的Rab35的可靠检测方法?方法:构建GST-RUN的原核表达载体,并使其在大肠杆菌BL21中大量表达?用GST pulldown和Western blot技术分别检测HEK293T细胞中活化的Rab35及Rab35蛋白的表达?结果:成功构建了GST-RUN的原核表达载体,并使其在原核细胞中大量表达融合蛋白GST-RUN,用GST Pulldown和Western blot技术证实了HEK293T细胞中有活化的Rab35和Rab35总蛋白的表达?结论:本工作所建立的GST Pulldown技术可以检测HEK293T细胞中活化的Rab35,从而为进一步深入研究Rab35在真核细胞中的功能提供了技术保障?

The activated Rab35 detected by GST pulldown in HEK293T cells

Objective:To establish a GST pulldown assay that can detect the activated Rab35 in eukaryotic cells. Methods:We constructed prokaryotic expression vector containing fusion protein GST-RUN. The correct plasmid was transfected into Ecoli. BL21 stain and the expression of the fusion protein was inducted by IPTG. SDS-PAGE and Western blotting were utilized for determination of the corresponding recombinant proteins. Then we pulled down the fusion protein with glutathione beads to detect the activated Rab35 in HEK293T cells. Results:The prokaryotic expression vector GST-RUN was successfully constructed and expressed fusion protein GST-RUN stably. GST pulldown assay showed high activated Rab35 in HEK293T cells. Conclusion:The activated Rab35 was successfully detected by GST pulldown assay. This experimental scheme can be used for further study of active Rab35 targeting in eukaryotic cells.