How to Detect Protein Ubiquitination-IP

Protein Ubiquitination is a crucial cellular biological process. It involves the binding of Ubiquitin protein to the target protein, thereby regulating the degradation, positioning, activation, etc., of the target protein. To detect protein ubiquitination, methods such as Immunoprecipitation (IP) combined with Western blot, mass spectrometry analysis, etc., are commonly used. Here are the typical steps for protein ubiquitination IP.

 

Analysis Workflow

1. Extraction of Cells or Tissue

Collect cell or tissue samples and lyse the samples with an appropriate cell lysis buffer (containing protease inhibitors) to release proteins.

 

2. Sample Pre-Treatment

Samples can be broken down by sonication or centrifugation to break cell nuclei. Add Dimethyl Sulfoxide (DMSO) or other cross-linking agents to stabilize protein-protein interactions.

 

3. Antibody Selection

Choose an antibody specific to the ubiquitination substrate you are interested in. Usually, anti-Ubiquitin antibody or a specific substrate antibody is used.

 

4. Immunoprecipitation (IP)

Bind the chosen antibody with Protein A/G agarose beads or magnetic beads to form an antibody-bead complex. Add the antibody-bead complex to the cell extract to allow the antibody to bind with the ubiquitination substrate. Rotate or shake the sample to allow the antibody to bind specifically to the ubiquitinated substrate.

 

5. Washing

Wash the antibody-bead complex multiple times with washing buffer to remove non-specifically bound proteins.

 

6. Protein Elution

Separate the precipitated protein using SDS-PAGE or other protein separation techniques.

 

7. Western Blot Analysis

First, transfer the separated proteins to a membrane. Use a specific antibody to detect the target protein to check the level of ubiquitination. A second antibody can be used to detect the antibody in the antibody-bead complex. Through Western blot analysis, the level of protein ubiquitination can be determined, such as the presence of ubiquitinated substrates and the level of ubiquitination.