Co-IP Detects Protein Ubiquitination

    Protein ubiquitination is a critical post-translational modification with significant implications in various biological processes, including the regulation of the cell cycle, gene transcription, and DNA repair. Co-immunoprecipitation (COIP) is a widely employed technique to assess protein ubiquitination.

     

    Protocol for COIP Detection Method

    1. Sample Preparation

    Utilize cell or tissue extracts as samples. Cells should be cultured under optimal conditions and treated 24 hours before the experiment.

     

    2. Protein Extraction

    Use a cell lysis buffer supplemented with ubiquitin protease inhibitors to avert the degradation of ubiquitinated proteins during the lysis process.

     

    3. Immunoprecipitation

    Employ a specific antibody targeting the protein of interest, mixing it with the protein extract. Introduce protein A or G agarose beads to facilitate binding, followed by gentle agitation. Subsequently, centrifugation is performed to isolate the antibody-antigen complexes from non-bound molecules.

     

    4. Protein Electrophoresis and Immunoblotting

    After precipitating protein samples, perform SDS-PAGE and transfer the proteins onto a membrane. Detect ubiquitinated proteins using an anti-ubiquitin antibody through immunoblotting.

     

    Considerations

    COIP demands precise antibodies and controlled experimental conditions, including pH, centrifugation speeds, and temperature. The selection and concentration of ubiquitin protease inhibitors should be carefully optimized to ensure reliable results. While COIP allows for qualitative analysis of protein ubiquitination, quantitative assessments necessitate additional methods, such as mass spectrometry.

     

    MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.

    Related Services

    Quantitative Ubiquitinomics Service

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