Application of HaloTag technology in the intracellular imaging and quantitative analysis of alpha-synuclein

doi:10.3969/j.issn.1006-7795.2014.03.013

Journal of Capital Medical University ›› 2014, Vol. 35 ›› Issue (3): 324-330.doi: 10.3969/j.issn.1006-7795.2014.03.013

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Application of HaloTag technology in the intracellular imaging and quantitative analysis of alpha-synuclein

Feng Xueting¹, Zhang Chenguang², Zhang Min², Yue Zhixia², Xing Tianyu², Li Shentao¹, Ding Wei²

1. Department of Immunology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China;
2. Department of Medical Genetics, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China

Received:2014-02-15 Online:2014-06-21 Published:2014-06-14
Supported by:
This study was supported by the National Natural Science Foundation of China(81372284, 81201816).

Abstract

Abstract:

Objective To develop a novel method for intracellular imaging and semi-quantification of alpha-synuclein(SNCA) using HaloTag technology by staining of fluorescent fusion proteins. Methods The mammalian expression vector of a Halo-SNCA was constructed, and the obtained plasmid was transfected into HEK293 cells. The expressed fusion protein was identified by Western blotting, and meanwhile labeled with TMR fluorescent ligand for laser confocal microscopy. The samples following imaging were subjected to protease digestion; the released ligands were quantified according to the measurements of fluorescence intensities to evaluate the expression levels of Halo-SNCA. Results The Halo-SNCA fusion protein was efficiently expressed in eukaryotic cells. The intracellular distribution of the Halo-SNCA was demonstrated by confocal microscopy with high resolution. Labeling with HaloTag fluorescent ligand was a practical approach to semi-quantitatively estimate the expression levels of Halo-SNCA with TMR fluorescent intensities. Conclusion The pHalo-SNCA vector can be a useful tool to analyze SNCA intracellular functions by simultaneously monitoring the localization/distribution and the dynamics of expression levels.

Key words: HaloTag, α-synuclein(SNCA), fluorescence imaging, fluorescence quantification

CLC Number:

R392.1

Feng Xueting, Zhang Chenguang, Zhang Min, Yue Zhixia, Xing Tianyu, Li Shentao, Ding Wei. Application of HaloTag technology in the intracellular imaging and quantitative analysis of alpha-synuclein[J]. Journal of Capital Medical University, 2014, 35(3): 324-330.

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Application of HaloTag technology in the intracellular imaging and quantitative analysis of alpha-synuclein

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