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Updated: Feb 6, 2026
Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method
Published on: July 27, 2016
Synthesis and purification of linkage-specific polyubiquitin chains of distinct length for structural studies
Anshumali Mittal1, Binita Shakya1
1Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, USA.
Abstract:
Polyubiquitylation is one of the most versatile post-translational modifications involved in the regulation of numerous intracellular signaling processes. An assembly procedure that is simple, robust, and efficient to synthesize and purify linkage-specific polyubiquitin chains of defined length at a preparative scale is required in biophysical and structural studies. Here, we have optimized known enzymatic procedures in the form of a protocol to obtain multi-milligrams of Lys48-and Lys63-linked polyubiquitin chain types with more than 99% purity. Mass spectrometry (ESI/MS) analysis of K48- and K63-linked diubiquitin confirmed that the enzymes used in the preparation generated homogeneous linkages with no promiscuity.
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