Assaying Arginylation Activity in Cell Lysates Using a Fluorescent Reporter.

ABSTRACT

Here, we describe an antibody-based method to evaluate the enzymatic activity of arginyltransferase1 (Ate1). The assay is based on the arginylation of a reporter protein, which contains the N-terminal peptide of beta-actin, a known endogenous substrate of Ate1, and a C-terminal GFP. The arginylation level of the reporter protein is determined  on an immunoblot with an antibody specific for the arginylated N-terminus, while the total amount of substrate is evaluated with anti-GFP antibody. This method can be used to conveniently and accurately examine the Ate1 activity in yeast and mammalian cell lysates. Moreover, the effect of mutation on Ate1 critical residues and effect of stress and other factors on Ate1 activity can also be successfully determined with this method.