Protein ubiquitination is an important cellular regulatory process that involves the chemical modification of ubiquitin protein binding to target proteins. This modification can modulate the stability, activity, subcellular localization, and interaction with other proteins of the target protein. To detect protein ubiquitination sites, some of the common methods can be taken:
1. Anti-ubiquitin antibody:
Using specific anti-ubiquitin antibodies, ubiquitinated proteins can be detected by western blotting or immunoprecipitation. These antibodies can recognize proteins associated with ubiquitination and thus determine which proteins are modified by ubiquitination.
2. Mass spectrometry analysis
Liquid chromatography-mass spectrometry (LC-MS MS) and multistage mass spectrometry (MS MS) techniques can be used to identify and quantify ubiquitinated proteins and their ubiquitination sites. This method typically requires purification of ubiquitinated proteins from cell extracts followed by mass spectrometry.
Samples were incubated with ubiquitin antibodies and antibodies to the protein of interest, and ubiquitination modifications were detected by PLA signal.
4. Ubiquitin-AMC enzyme activity determination
This is an in vitro method used to determine the activity of protein ubiquitinating enzymes. This method uses a substrate with carbamoyl hydroxyaminomethyl ketone (Ubiquitin-AMC) to determine the activity of ubiquitinase by monitoring the substrate degradation rate.
5. Bioinformatics Tools:
A number of bioinformatics tools and databases can be used to ** potential ubiquitination sites. These tools use different algorithms and data to determine which proteins are likely to be ubiquitinated, and the likelihood of ubiquitination sites.