Protocol

Split Luciferase Complementation Assay for Studying Interaction of Proteins X and Y in Cells

This protocol was adapted from "Imaging Protein-Protein Interactions in Living Animals," Chapter 35, in Protein-Protein Interactions (ed. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

This protocol describes a split luciferase complementation assay used to study the interaction of proteins in cells. In the "split protein" strategy, a single reporter protein/enzyme (in this case, firefly luciferase [Fluc]) is cleaved into amino-terminal and carboxy-terminal halves; each half is fused to one of two interacting proteins, X and Y. Physical interactions between the two proteins, X and Y, reconstitute the functional reporter protein, leading to enzymatic activities that can be measured by in vitro or in vivo assays. It is important to perform the assay initially in cell culture before proceeding with animal imaging, not only to conserve animals, but also to establish the sensitivity of the assay.