Constructing and Expressing GFP Fusion Proteins

Spector, D. L., Goldman, R. D. (2006) Constructing and Expressing GFP Fusion Proteins. Cold Spring Harbor Protocols, 2006 (7). ISSN 1940-3402 (Print) 1559-6095 (Electronic)

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URL: http://www.ncbi.nlm.nih.gov/pubmed/22484672
DOI: 10.1101/pdb.prot4649

Abstract

INTRODUCTION GFP (green fluorescent protein) fusion proteins have been used to address a wide range of questions in individual cells, as well as in tissues of a particular organism. GFP fusion proteins can be transiently or stably expressed. Although transient expression is quick and can provide informative results, in many cases it is beneficial and/or essential to develop stable cell lines expressing the fusion protein of interest. In addition to providing more native levels of expression, individual clones can be generated from single cells, the integration site of the plasmid mapped, and the copy number determined. Because every cell in the population is expressing the fusion protein, cell cycle analyses and biochemical fractionation are significantly easier to accomplish. The following protocol can be used for the development of stable cell lines expressing GFP fusion proteins. Although optimal transfection procedures (e.g., calcium phosphate, electroporation, or FuGENE 6 [Roche Applied Science]) vary depending on cell type, this general transfection procedure has been successful for stable transfection of HeLa, A-431, U2OS, BHK, and HT1080 cells.

Item Type: Paper
Subjects: Investigative techniques and equipment
Investigative techniques and equipment > cell fusion
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > green fluorescent protein
CSHL Authors:
Communities: CSHL labs > Spector lab
Depositing User: Matt Covey
Date Deposited: 10 Dec 2012 20:02
Last Modified: 28 Jan 2015 21:30
Related URLs:
URI: http://repository.cshl.edu/id/eprint/26359

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