Assaying Intracellular Arginylation Activity Using a Fluorescent Reporter

Abstract

In this chapter, we present a simplified version of the method described in Chapter 9 of this book, adapted for fast and convenient evaluation of intracellular arginylation activity in live cells. As in the previous chapter, this method utilizes a GFP-tagged N-terminal β-actin peptide transfected into cells as a reporter construct. Arginylation activity can then be evaluated by harvesting the reporter-expressing cells and analyzing them directly by Western blot using an arginylated β-actin antibody and a GFP antibody as an internal reference. While absolute arginylation activity cannot be measured in this assay, different types of reporter-expressing cells can be directly compared, and the effect of genetic background or treatment can be evaluated. For its simplicity and broad biological application, we felt this method merited presentation here as a separate protocol.