Protocol

Subcellular Localization of GUS- and GFP-Tagged Proteins in Onion Epidermal Cells

This protocol was adapted from "How to Study Gene Expression," Chapter 7, in Arabidopsis: A Laboratory Manual (eds. Weigel and Glazebrook). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2002.

INTRODUCTION

Recombinant tags (i.e., reporter proteins) offer an excellent alternative to antibodies for determining the subcellular localization of proteins. The most user-friendly tags are the ß-glucuronidase (GUS) reporter enzyme from Escherichia coli and fluorescent proteins derived primarily from the green fluorescent protein (GFP) of the jellyfish Aequorea victoria. GUS is useful primarily as a tag to address nuclear localization, whereas GFP is more versatile. Moreover, GFP is detectable directly in living cells, whereas GUS is only detected indirectly by staining of fixed tissue. This may lead to artifacts or it may obscure problems with protein solubility. In this protocol, protein localization is routinely assayed after particle-mediated transient transformation of onion epidermal cells. With this method it can be determined rapidly whether a given fusion protein is active, and preliminary targeting data can be obtained.