Universität zu Köln

Schuster, Ramona ORCID: 0000-0002-2227-3718, Simoes, Tania, den Brave, Fabian ORCID: 0000-0001-6955-4616 and Escobar-Henriques, Mafalda ORCID: 0000-0002-0879-3119 (2018). Separation and Visualization of Low Abundant Ubiquitylated Forms. Bio-protocol, 8 (22). SUNNYVALE: BIO-PROTOCOL. ISSN 2331-8325

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Abstract

In this protocol we describe the separation and visualization of ubiquitylated forms of the yeast mitofusin Fzo1 by Western blot. To this aim, we express HA-tagged Fzo1 in Saccharomyces cerevisiae, break the cells to extract a membrane-enriched fraction, solubilize the membranes using detergent and then specifically immunoprecipitate the tagged protein using anti-HA affinity beads. Subsequently, we separate the higher molecular weight (ubiquitylated) forms of Fzo1 via SDS-PAGE. Finally, immunoblotting and immunodecoration are used to detect the protein and its ubiquitylated forms using an HA-specific antibody. By using this protocol, it is possible to separate and visualize higher molecular weight forms of low abundant proteins such as Fzo1 and detect sharp and distinct bands above the unmodified protein by Western blot.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Schuster, Ramona UNSPECIFIED orcid.org/0000-0002-2227-3718 UNSPECIFIED Simoes, Tania UNSPECIFIED UNSPECIFIED UNSPECIFIED den Brave, Fabian UNSPECIFIED orcid.org/0000-0001-6955-4616 UNSPECIFIED Escobar-Henriques, Mafalda UNSPECIFIED orcid.org/0000-0002-0879-3119 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-165529
DOI:	10.21769/BioProtoc.3081
Journal or Publication Title:	Bio-protocol
Volume:	8
Number:	22
Date:	2018
Publisher:	BIO-PROTOCOL
Place of Publication:	SUNNYVALE
ISSN:	2331-8325
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Institute for Genetics
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language Biology Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/16552