Protocol

Introduction of Caged Peptide/Protein into Cells Using Microinjection

This protocol was adapted from "Application of Light-Directed Activation of Caged Biomolecules and CALI to Problems in Cell Motility," Chapter 10, in Live Cell Imaging (eds. Goldman and Spector). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

Activation and inactivation of proteins using photoactivation of caged peptides or proteins offer insights into cellular dynamics not achievable using genetic means. The ability to selectively alter the activity of a specific protein at a defined time and location inside a cell allows the correlation of changes in protein activity and cellular behavior. A caged compound, peptide, or protein is prepared by covalently linking it to a photolabile, protecting group via a limited number of critical functional groups in the biomolecules. Under carefully defined conditions, a brief irradiation with UV light (320-400 nm) can lead to an intramolecular photoisomerization reaction that induces the cleavage of the chemical bond between the cage group and the unmodified, biologically active species. The following protocol uses a semiautomated microinjection system (Eppendorf FemtoJet Microinjector along with the Eppendorf InjectMan) for the introduction of caged compounds into cells. Unlike the bead-loading approach, this technique allows the investigator to choose which cells are targeted.