How to Detect the Ubiquitination Level of a Protein

Ubiquitination is a widely present method of protein modification. It manages its designation, activity, stability, and interaction by marking proteins, playing a crucial role in the regulation of cellular functions and biological processes. Therefore, measuring the ubiquitination level of a protein is significant for understanding its function and regulation mechanism. This article mainly introduces how to detect the ubiquitination level of a protein.

 

Immunoblotting

Immunoblotting is a common method for detecting the level of protein ubiquitination, mainly through the detection of ubiquitinated proteins in protein samples using specific antibodies. Firstly, the protein sample is separated by SDS-PAGE, then the protein is transferred to a Polyvinylidene Fluoride (PVDF) membrane, followed by incubation with anti-ubiquitin antibodies. Finally, the presence of ubiquitinated proteins is determined by detecting the labeled antibodies.

 

Mass Spectrometry

Mass spectrometry can provide more detailed and accurate information, including ubiquitination sites and types of ubiquitin chains. Firstly, the protein sample needs to be enzymatically digested, and then the ubiquitination site is analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Finally, the ubiquitinated proteins and their ubiquitination sites can be determined by advanced mass spectrometry data analysis (such as database search and protein quantitative analysis).

 

Immunoprecipitation

Immunoprecipitation is a technique for detecting interactions between proteins and can also be used to detect the level of protein ubiquitination. This method, using the specificity of antibodies, can enrich specific ubiquitinated proteins. Firstly, the protein sample is incubated with a specific antibody, then the antibody-antigen complex is enriched with protein A/G magnetic beads, and finally, the level of protein ubiquitination is detected by immunoblotting.

 

Immunofluorescence

Immunofluorescence is a method for detecting the distribution and location of proteins in cells or tissues and can also be used to detect the level of protein ubiquitination. This method, using fluorescently labeled antibodies, can directly observe the distribution and location of ubiquitinated proteins in cells or tissues.

 

Before performing the above experiments, ensure that the antibodies used have high specificity and high affinity, and the treatment of samples (such as washing, separation, and concentration) should be strictly carried out according to the experimental procedure to prevent false-positive or false-negative results.