Polyubiquitination Detection

Ubiquitination is a unique post-translational modification process that involves attaching a small protein of 76 amino acids, known as ubiquitin, to the target protein one by one through a three-enzyme cascade reaction (E1, E2, and E3 enzymes). This process can be repeated to form a ubiquitin chain on the ubiquitinated target protein, a phenomenon known as polyubiquitination. Polyubiquitination plays important roles in many biological processes, including protein degradation, immune response, DNA repair, cell cycle regulation, and so on. Therefore, the detection and quantification of polyubiquitination are crucial for understanding these processes.

 

Methods for Detecting Polyubiquitination

1. Western Blot Analysis

The target protein is extracted from the cell or tissue sample and then separated by SDS-PAGE electrophoresis. Then, the separated proteins are transferred to a membrane and immunoblotted with ubiquitin-specific antibodies. Finally, the degree of polyubiquitination is observed and quantified by fluorescence or chemiluminescence detection.

 

2. Immunoprecipitation (IP)

Immunoprecipitation is another commonly used method for detecting polyubiquitination. Ubiquitinated proteins are precipitated from cell samples using ubiquitin-specific antibodies. Then, SDS-PAGE electrophoresis and immunoblotting are used to detect and quantify polyubiquitin chains.

 

3. Mass Spectrometry

Mass spectrometry can directly identify ubiquitination sites and obtain precise structural information of ubiquitin chains. The ubiquitinated proteins or polyubiquitin chains are extracted and purified from the cell sample. Then, proteins are cut by protease or proteinase K and the information on ubiquitination sites and chain structure is obtained by liquid chromatography-mass spectrometry analysis.

 

Polyubiquitination is an important post-translational modification process that forms ubiquitin chains by attaching ubiquitin to target proteins, playing important roles in biological processes such as protein degradation, immune response, DNA repair, cell cycle regulation, and so on. Common methods for detecting polyubiquitination include Western blot analysis, immunoprecipitation, and mass spectrometry. By comprehensively utilizing these methods, the function and regulatory mechanisms of polyubiquitination in biological processes can be revealed.