Published: Vol 8, Iss 22, Nov 20, 2018 DOI: 10.21769/BioProtoc.3081 Views: 4758
Reviewed by: Jyotiska ChaudhuriAlvaro Eduardo GalvisMario Valentino
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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.
Keywords: Fzo1Background
Immunoprecipitation is a method to precipitate and enrich a protein from an extract by using an antibody specifically binding to it. The enrichment of a protein is of particular importance when analyzing lower abundant modified forms of it, as is usually the case for ubiquitylated forms of a protein. To be able to enrich for the ubiquitylated Fzo1 protein we tagged the protein with a Hemagglutinin (HA) epitope and use anti-HA affinity beads to precipitate this modified protein from the solubilized membrane fraction. This allows for the detection of the less abundant ubiquitylated forms of proteins as for example Fzo1 (Anton et al., 2011 and 2013; Simoes et al., 2018).
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Acknowledgments
Funding: Deutsche Forschungsgemeinschaft (ES338/3-1); Universität zu Köln (German Excellence Initiative and Faculty of Mathematics and Natural Sciences); Deutsche Forschungsgemeinschaft (SFB635); Deutsche Forschungsgemeinschaft (CRC1218TPA03).
Competing interests
The authors declare they have no conflict of interest or competing interests.
References
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Copyright
Schuster et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
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Category
Biochemistry > Protein > Immunodetection
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