Ubiquitin is a highly conserved polypeptides, which is formed by 76 amino acid residues. It can covalently connect with one or more lysine residues on the intracellular target protein under the condition of enzymatic reactions catalyze. Ubiquitin itself contains seven lysine residues, which can also connect to each other to form a multi-ubiquitin chains (polyubiquitin chain). The present study shows that if the first 48 lysine residues polyubiquitin chain and connected to the modified protein will mediate target protein into the proteasome and are degraded; if the protein is modified with other sites, such as the first 63 lysine residues connected to the target protein signaling pathway function can play without being degraded.
Approaches for detecting ubiquitinated proteins
Before this detection, we should firstly know following three points:
1. Proteins, which have been ubiquitinated.
2. Lysine residue, which occurred ubiquitination.
3. Ubiquitination prediction
Then the aims for this detection are that what molecular effects are produced by ubiquitinated proteins and its effect on downstream signaling pathways.
The methods and processes of content above:
Western blot and strip
Detecting ubiquitinated protein band with western blot and stripping the membrane, which should be recorded with pictures. And then it should react with specific proteins antibodies and specific ubiquitination sites antibodies, whose color will be recorded. By comparing the positive bands to judge the occurrence of protein ubiquitination of a specific site preliminarily.
Western blot and immunoprecipitation
To seperate the special proteins and proteins, which has been combined with the special proteins by using co-immunoprecipitation. And then separated proteins should be analyzed by SDS and western blot. This method can make it clear that which lysine residue of certain protein has occurred ubiquitylation.
In vitro ubiquitination assay
Transfecting the target genes with 293 cells to make it express abundantly. And then target proteins will be extracted in 24 hours. In vitro reaction buffer, we should incubate ubiquitinated proteins and protein causing protein A ubiquitination with UBE1, UbeH13-Uev 1 a heterodimer complex and HA-ubiquitin. The products should be analyzed by IP and WB. This method can show the E3 ligase, which cause the proteins causing the protein ubiquitination.
In vitro ubiquitin-binding assay
Transfecting target proteins with 293 cells. And purifying it after its abundant expression or extracting endogenous proteins directly. And then incubating it with K63- or K48-linked ubiquitin chain petides in vitro. After that is should be detected with SDS and ubiquitin antibodies. This method is used to determine lysine residues, which are easy to be ubiquitinated.
These are four methods for detecting protein ubiquitination.
References
http://www.creative-proteomics.com/services/Ubiquitination.htm
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