How Is Ubiquitinated Protein Generally Detected
Ubiquitination is a post-translational modification process in which ubiquitin is attached to proteins, regulating their degradation, signaling, and other functions. Common methods for detecting ubiquitinated proteins include:
1. Western Blot (WB)
Protein extraction is performed on cell or tissue samples, followed by SDS-PAGE separation. The separated proteins are transferred to a membrane and incubated with an anti-ubiquitin antibody. If ubiquitination occurs, higher molecular weight ubiquitinated protein bands can be observed. To detect ubiquitination of specific proteins, specific antibodies against the target protein can be used.
2. Immunoprecipitation (IP)
Anti-ubiquitin or specific protein antibodies are combined with protein A/G magnetic beads, and cell or tissue lysates are incubated with the beads. Through magnetic separation, ubiquitinated proteins or ubiquitinated forms of specific proteins can be precipitated. The precipitate is then analyzed by SDS-PAGE and Western Blot to detect ubiquitination.
3. Mass Spectrometry (MS)
A highly sensitive and high-throughput method for detecting ubiquitinated proteins. Proteins are extracted, digested with enzymes, and analyzed using liquid chromatography-mass spectrometry (LC-MS/MS). Mass spectrometry data can identify ubiquitination sites and quantify ubiquitination levels.
4. Ubiquitination Enzyme Activity Assay
Ubiquitination can be indirectly detected by measuring the activity of ubiquitination enzymes (such as E1, E2, and E3). Enzyme activity assays typically use fluorescence, luminescence, or colorimetric-based in vitro assay kits.
5. Yeast Two-Hybrid (Y2H)
This technique is used to screen proteins interacting with ubiquitination enzymes or ubiquitin-binding proteins, revealing potential ubiquitination targets. By fusing the target protein to the activation and DNA-binding domains in the yeast two-hybrid system, interacting protein pairs in yeast can be identified.
6. Bioinformatics Analysis
Computational methods can predict ubiquitination sites and potential ubiquitinated proteins. By analyzing protein sequence conservation across species, potential ubiquitination sites can be identified. Additionally, protein networks and signaling pathways can be examined to predict biological processes involving ubiquitination.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
Related Services
How to order?
