Protein Detection for Ubiquitination
Ubiquitinated protein detection is to identify and quantify the status of proteins after ubiquitination post-translation modification. Here are some commonly used methods to detect ubiquitinated proteins:
1. Western Blot Analysis
Western blotting is a common method for detecting ubiquitinated proteins. This process usually involves:
(1) Extracting protein samples.
(2) Separating proteins using SDS-PAGE electrophoresis.
(3) Transferring proteins onto a membrane.
(4) Using antibodies against ubiquitin for detection, to identify ubiquitinated proteins.
2. Immunoprecipitation (IP)
(1) Purpose: To enrich specific ubiquitinated target proteins.
(2) Method: Use specific antibodies of the target protein for immunoprecipitation, followed by Western blotting, using anti-ubiquitin antibodies to detect ubiquitination.
3. Tandem Immunoprecipitation
(1) Purpose: To identify specific types of ubiquitin chains associated with the target protein.
(2) Method: First use an antibody against a specific type of ubiquitin chain (e.g., anti-K48 or anti-K63 chain antibodies) for precipitation, followed by a second precipitation using an antibody for the target protein.
4. Mass Spectrometry
(1) Purpose: To identify ubiquitination sites and types of ubiquitin chains.
(2) Method: Use LC-MS/MS technology to identify ubiquitinated proteins and modification sites. Often combined with enzymatic digestion (such as Trypsin digestion) to produce peptides, which are then analyzed.
5. Fluorescence Microscopy
Using fluorescent labels to mark ubiquitin, observe the location and distribution of ubiquitinated proteins in cells. This method can be used to monitor ubiquitination events in real time.
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