Lin Michael Z, Tsien Roger Y
Department of Pediatrics and Programs in Gene Therapy and Molecular Imaging, Stanford University, Stanford, California, USA.
Curr Protoc Protein Sci. 2010 Feb;Chapter 26:Unit 26.5. doi: 10.1002/0471140864.ps2605s59.
The ability to quantify or visualize newly synthesized proteins has important uses in cell biology. For example, a researcher may wish to quantify basal or inducible rates of translation of a specific gene of interest, or detect subcellular locations of newly synthesized copies of a protein in order to study the role of new protein synthesis in the growth of specialized cellular structures. In this unit, the TimeSTAMP method for labeling of newly synthesized copies of a protein of interest is described. In the TimeSTAMP method, the experimenter expresses a protein of interest as a fusion with a cis-acting protease and an epitope tag, both of which are removed by default protease activity. Addition of a specific protease inhibitor then allows preservation of the tag on subsequently synthesized proteins. Finally, the tag is detected by immunological methods.
对新合成蛋白质进行定量或可视化的能力在细胞生物学中具有重要用途。例如，研究人员可能希望定量特定感兴趣基因的基础翻译率或诱导翻译率，或者检测蛋白质新合成拷贝的亚细胞定位，以便研究新蛋白质合成在特殊细胞结构生长中的作用。在本单元中，将描述用于标记感兴趣蛋白质新合成拷贝的TimeSTAMP方法。在TimeSTAMP方法中，实验者将感兴趣的蛋白质表达为与顺式作用蛋白酶和表位标签的融合蛋白，默认的蛋白酶活性会将二者都去除。添加特定的蛋白酶抑制剂可使标签保留在随后合成的蛋白质上。最后，通过免疫学方法检测该标签。
