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Citation: CUI Xiu-Yun, SUN Ning-Ning, XIE Xiao-Na, SUN Wan-Chun, ZHAO Qing, LIU Ning. Detection of Newly Synthesized Proteins via Metabolic Incorporation of Non-Natural Amino Acid[J]. Chinese Journal of Analytical Chemistry, ;2018, 46(11): 1808-1813. doi: 10.11895/j.issn.0253-3820.171035 shu

Detection of Newly Synthesized Proteins via Metabolic Incorporation of Non-Natural Amino Acid

¹ Central Laboratory, The Second Hospital, Key Laboratory of Zoonosis, Ministry of Education, Jilin University, Changchun 130041, China;
² Department of Endocrinology, The First Hospital of Jilin University, Changchun 130012, China;
³ Department of Neurology, China-Japan Union Hospital, Changchun 130033, China

Received Date: 26 October 2017
Revised Date: 25 June 2018

Fund Project: This work was supported by the National Natural Science Foundation of China (Nos.81472030), the Jilin Province Science and Technology Department (Nos. 20110739, 20180101267JC), the Jilin University Bethune Project B (No. 2012210) and the Health and Family Planning Commission of Jilin Province (No. 2015Z041).

A method for detection of newly synthesized protein via metabolic incorporation of non-natural amino acid was developed, in which Raw264.7 cells were treated by lipopolysaccharide (LPS) to generate tumor necrosis factor alpha (TNF-α). Experimental parameters including the concentration of LPS and the duration of LPS treatment were investigated by measuring the LPS stimulated TNF-α from Raw264.7 cells. The optimal experimental conditions were determined as follows. Raw264.7 cells were cultured in Met-free DMEM containing 1% fetal bovine serum (FBS), and then treated for 4 h with LPS at concentration of 10 ng/mL in presence of AHA. The biotin tags were introduced into proteins containing azidohomoalanine (AHA) by copper-catalyzed alkyne-azidecycloaddition (CuAAC). Then TNF-α was specifically captured by the antibody adsorbed on the solid support, on which the biotin tag could be detected by streptavidin coupled with horseradish peroxidase (HRP). This study provided a method for the detection of a specific newly synthesized protein, and characterization of specific protein turnover.
- Newly synthesized proteins,
- Non-natural amino acid,
- Tumor necrosis factor α,
- Click chemistry
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