Subcellular Localization of Intracellular Human Proteins by Construction of Tagged Fusion Proteins and Transient Expression in COS-7 Cells
Identifying the subcellular compartment of a protein is an important step toward assigning protein function. Starting with a clone containing the open reading frame (ORF) of interest, it is possible to attach a variety of short amino acid tags or fluorescent proteins and detect the location of the protein, after transfection into a cell line, using fluorescent microscopy. By collecting data from various expression clone constructs, using a range of cell lines and double labeling with cellular compartment markers, a picture of the localization of a gene can be built up. This chapter describes how to obtain the ORF clone for your gene of interest, clone it into your choice of mammalian expression vector or vectors, transiently transfect for visualization, and where to get started when interpreting the results.
Keywordssubcellular localization open reading frame mammalian expression clone fluorescent microscopy
We thank Begoña Aguado, Carol Edwards, Catherine Taylor, Charmain Wright, Frida Andersson, Gözde Akdeniz, James Grinham, Matthew Davis, and Meera Mallya for assistance with developing the protocols. We also thank Catherine Taylor and Ian Dunham for critically reading the manuscript. This work was supported by the Wellcome Trust.