ArticleMethods in molecular biology (Clifton, N.J.)2026
Synthesis of Ub-ACA, a Fluorogenic DUB Substrate via ACPL Method.
Article in Methods in molecular biology (Clifton, N.J.), 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
This chapter details the step-by-step synthesis of ubiquitin-7-amino-4-coumarinyl-acetic acid (Ub-ACA) using the activated cysteine-directed protein ligation (ACPL) technique. Ub-ACA is a fluorogenic substrate for deubiquitinases (DUBs). It can be readily synthesized via ACPL to a high quantity by exchanging a C-terminal cysteine residue in a recombinantly expressed Ub-G76C mutant with Gly-ACA, a process mediated simply by 2-nitro-5-thiocyanatobenzoic acid (NTCB). This chapter provides protocols for protein expression, thiol activation, fluorophore conjugation, and product purification, yielding Ub-ACA with high purity suitable for enzymatic assays.
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