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Methods Mol Biol. 2016;1475:109-21. doi: 10.1007/978-1-4939-6358-4_8.

Using Biotinylated SUMO-Traps to Analyze SUMOylated Proteins.

Author information

1
Ubiquitylation and Cancer MolecularBiology (UMCB) Laboratory, Inbiomed, 20009, San Sebastian-Donostia, Gipuzkoa, Spain.
2
Group of Ubiquitin-Dependent Proteolysis and Intracellular Communication (G(u)ic), Institute of Biomedical Imaging and Life Sciences (IBILI), University of Coimbra, 3000-548, Coimbra, Portugal.
3
CIC bioGUNE, Bizkaia Technology Park, 48160, Derio, Bizkaia, Spain.
4
INBIOMED, San Sebastian, Spain. manuel.rodriguez@itav.fr.

Abstract

SUMO-interacting motifs (SIMs) recognize SUMOylated proteins with high specificity allowing to connect SUMO-modified proteins. Multiple SIMs fused to distinct tags have been used to increase their affinity and generate more efficient purification tools. Enrichment of SUMOylated proteins using SIMs arranged in tandem (SUMO-traps) facilitates the identification and characterization of protein targets in vitro and in vivo. Here a protocol to produce biotinylated SUMO-traps (bioSUBEs) to capture SUMO chains and typical SUMOylated proteins such as p53 or IkB╬▒ is presented. Biotinylated SUMO-traps represent an alternative to reduce the background associated to bigger tags, e.g., during mass spectrometry analysis. Consequently, bioSUBEs are alternative tools to characterize endogenous SUMO targets.

KEYWORDS:

Analysis; Biotinylation; BirA; Purification; SIMs; SUBEs; SUMOylation

PMID:
27631801
DOI:
10.1007/978-1-4939-6358-4_8
[Indexed for MEDLINE]

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