Suspension Bead Loading (SBL): An Economical Protein Delivery Platform to Study URM1’s Behavior in Live Cells

Abstract Uniquely modified synthetic proteins are difficult to produce in large quantities, which could limit their use in various in vitro settings and in cellular studies. In this study, we developed a method named “suspension bead loading” (SBL), to deliver protein molecules into suspended living cells using glass beads, which significantly reduces the amount of protein required for effective delivery. We investigated the delivery efficiency of functionally different proteins and evaluated the cytotoxic effect of our method and the chemical and functional integrity of the delivered protein. We utilized SBL to address questions related to ubiquitin‐related modifier 1 (URM1). Employing minimal protein quantities, SBL has enabled us to study its behavior within live cells under different redox conditions, including subcellular localization and conjugation patterns. We demonstrate that oxidative stress alters both the localization and conjugation pattern of URM1 in cells, highlighting its possible role in cellular response to such extreme conditions.