Separation and Visualization of Low Abundant Ubiquitylated Forms

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Mitofusins, like Fzo1 in yeast, are crucial for mitochondrial fusion.
• Ubiquitylation is a key post-translational modification regulating protein function and stability.
• Detecting ubiquitylated forms of low-abundance proteins presents a technical challenge.

Purpose of the Study:

• To establish a robust protocol for the separation and visualization of ubiquitylated Fzo1.
• To enable the study of Fzo1 ubiquitylation in *Saccharomyces cerevisiae*.

Main Methods:

• Expression of hemagglutinin (HA)-tagged Fzo1 in yeast (*Saccharomyces cerevisiae*).
• Isolation of a membrane-enriched fraction and solubilization using detergents.
• Immunoprecipitation of HA-tagged Fzo1 using anti-HA affinity beads.
• Separation of ubiquitylated forms by SDS-PAGE and detection via immunoblotting with an HA-specific antibody.

Main Results:

• Successful separation and visualization of higher molecular weight ubiquitylated forms of Fzo1.
• Detection of distinct bands corresponding to ubiquitylated Fzo1 above the unmodified protein.
• Demonstration of the protocol's efficacy for low-abundance proteins.

Conclusions:

• The described Western blot protocol effectively visualizes ubiquitylated Fzo1.
• This method facilitates the study of ubiquitylation in mitochondrial dynamics.
• The protocol is applicable to other low-abundance proteins.