High-throughput methods for the detection of protein overexpression using fluorescence markers

Gabrielsen, M. , Roe, A.J. and McLuskey, K. (2013) High-throughput methods for the detection of protein overexpression using fluorescence markers. Methods in Cell Biology, 113, pp. 189-208. (doi: 10.1016/B978-0-12-407239-8.00010-0)

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.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Gabrielsen, Dr Mads and McLuskey, Dr Karen and Roe, Professor Andrew
• Authors: Gabrielsen, M., Roe, A.J., and McLuskey, K.
• College/School: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Journal Name: Methods in Cell Biology
• Publisher: Academic Press
• ISSN: 0091-679X