Common Methods for Protein SUMOylation Detection

Protein SUMOylation is a vital post-translational modification process, involving the covalent binding of small ubiquitin-like modifier (SUMO) to the target protein. SUMOylation plays a crucial role in many biological processes, such as nuclear transport, transcriptional regulation, cell cycle progression, and stress response. Here are the commonly used methods to detect protein SUMOylation:

 

1. Western Blot

Utilizing specific anti-SUMO antibodies, it is possible to directly detect whether the protein has undergone SUMOylation modification. SUMOylation usually results in an increase in protein molecular weight, which can be observed after electrophoretic separation.

 

2. Immunoprecipitation (IP) + Western Blot

Firstly, the target protein is precipitated from the cell lysate using a specific antibody, then Western Blot with anti-SUMO antibody is used to detect its SUMOylation status.

 

3. Affinity Purification Followed by Mass Spectrometry

Through the affinity purification of SUMO binding sites, SUMOylated proteins can be enriched from complex samples, then identified using mass spectrometry.

 

4. Bioluminescence Resonance Energy Transfer (BRET)

Detects the dynamic process of SUMOylation in cells. It usually involves linking fluorescent proteins and luciferase tags to the target protein and SUMO respectively, and then measuring the energy transfer between them to detect their interaction.

 

5. Confocal Fluorescence Microscopy

With fluorescently labeled SUMO and target protein, the location and dynamics of SUMOylation modification can be directly observed in cells.

 

6. Enzyme-Linked Immunosorbent Assay (ELISA)

Using specific anti-SUMO antibodies, quantitative analysis of SUMOylated proteins can be performed.