title: High-throughput methods for the detection of protein overexpression using fluorescence markers.
abstract: Overexpression of recombinant protein is essential for the structural or biochemical characterization of a protein, and the aim is to obtain as much correctly folded protein as possible. The use of fluorescence markers such as green fluorescent protein (GFP) to monitor protein overexpression has been established for a number of years, where the fluorescent signal is a direct measure of protein quantity and quality. In this protocol, GFP is used to quickly identify the optimal overexpression conditions for six individual protein targets, by testing four growth media, four cell lines, and two temperatures. Common examples are suggested in the text, but can be replaced by the user. Methods to visualize the fluorescence readings as a heat-map, and to perform dot-blot analysis using an antibody against common tags are also described.
authors: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.
authors: Mads
authors: M
authors: Gabrielsen
authors: Andrew J
authors: AJ
authors: Roe
authors: Karen
authors: K
authors: McLuskey
mesh_terms: D003001:Cloning, Molecular; D003470:Culture Media; D046508:Culture Techniques; D004926:Escherichia coli; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012995:Solubility; D014169:Transformation, Bacterial
chemical_list: D003470:Culture Media; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins