GST pulldown技术检测HEK293T细胞Daam1的活性

目的:运用GST pulldown技术建立真核细胞中Daam1活性检测的可靠方法?方法:构建GST-RhoA的原核表达载体,并使其在大肠杆菌BL21菌株中大量表达?用GST pulldown和Western blot技术分别检测HEK293T细胞中活化的Daam1及Daam1蛋白的表达?结果:在成功构建了GST-RhoA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-RhoA后,用GST pulldown和Western blot技术证实HEK293T细胞中有活化的Daam1和Daam1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Daam1的活性,从而为进一步深入研究Daam1在真核细胞中的功能提供了技术保障?

The activity of Daam1 detected by GST pulldown in HEK293T cells

Objective: To establish a GST pulldown assay for detection of Daam1 activity in eukaryotic cells. Methods: Prokaryotic expression vector containing fusion protein GST-RhoA was constructed. The correct plasmid was transfected into E. coli. BL21 stain and inducted the expression of the fusion protein by IPTG. SDS-PAGE and Western blotting were utilized to determine the corresponding recombinant proteins. The fusion protein with glutathione beads was pulled down to detect the activity of Daam1 in HEK293T cells. Results: GST-RhoA vector was successfully conducted and stably expressed fusion protein GST-RhoA. The activity of Daam1 was detected by GST pulldown assay,showing high activity of Daam1 in HEK293T cells. Conclusion: The activity of Daam1 was successfully detected by GST pulldown assay. This experimental scheme can be used for further studies of active Daam1 targeting in eukaryotic cells.