Analysis of Lysine Acetylation and Acetylation‐like Acylation In Vitro and In Vivo

Abstract Protein lysine acetylation refers to the covalent transfer of an acetyl moiety from acetyl coenzyme A to the epsilon‐amino group of a lysine residue and is critical for regulating protein functions in almost all living cells or organisms. Studies in the past decade have demonstrated the unexpected finding that acetylation‐like acylation, such as succinylation, propionylation, butyrylation, crotonylation, and lactylation, is also present in histones and many non‐histone proteins. Acetylation and acetylation‐like acylation serve as reversible on/off switches for regulating protein function while interplaying with other post‐translational modifications (such as phosphorylation and methylation) in a codified manner. Lysine acetylation and acetylation‐like acylation are important for regulating different cellular and developmental processes in normal and pathological states. Thus, the detection of such modifications is important for related basic research and molecular diagnostics. Traditionally, lysine acetylation is detected by autoradiography, but recent decades have seen great improvement in the quality of site‐specific antibodies against acetylation (or acetylation‐like acylation), thereby providing competitive alternatives to the use of radioactive acetate and acetyl‐coenzyme A for in vivo and in vitro labeling, respectively. This article describes protocols for the detection of lysine acetylation and acetylation‐like acylation with site‐specific antibodies to complement extant autoradiography‐based methods (Pelletier et al., 2017). © 2023 Wiley Periodicals LLC. Basic Protocol 1 : Acylation assays in vitro Basic Protocol 2 : Determination of in vivo acylation