How Should Endogenous SUMO Co-IP Be Performed
Endogenous Co-Immunoprecipitation (Co-IP) of SUMO (Small Ubiquitin-like Modifier)–modified proteins is typically conducted according to the following steps:
1. Cell Collection
Begin by harvesting cells. For studies of SUMOylation, transiently transfected cells or cells subjected to specific treatments are commonly used to enhance the levels of SUMOylated proteins.
2. Preparation of Lysis Buffer
Prepare a lysis buffer containing protease inhibitors and deSUMOylation inhibitors, such as N-ethylmaleimide (NEM). NEM effectively inhibits SUMO-specific proteases (deSUMOylases), thereby preserving SUMO modifications.
3. Cell Lysis
Lyse the harvested cells in the prepared lysis buffer. This step should be performed at low temperature (e.g., on ice) to prevent proteolysis and deSUMOylation.
4. Centrifugation
Centrifuge the lysates at high speed to pellet cell debris and obtain the clarified supernatant.
5. Pre-Clearing of Lysates
Pre-clear the supernatant to remove proteins that bind non-specifically, typically by incubating with protein A/G agarose beads or with isotype-matched control antibodies.
6. Antibody Incubation
Add a specific antibody against the target protein to the pre-cleared lysate to capture its SUMOylated forms. Alternatively, antibodies directed against SUMO can be used directly to enrich SUMO-modified proteins.
7. Overnight Incubation
Incubate the mixture overnight at 4°C to ensure efficient binding between the antibody and the target protein.
8. Capture of Immune Complexes
Add protein A/G agarose beads to bind the antibody-protein complexes.
9. Washing of Beads
Wash the beads several times with lysis buffer or a designated wash buffer to eliminate non-specifically bound proteins.
10. Elution of Immune Complexes
Elute the immune complexes by adding SDS-PAGE sample loading buffer and heating the samples.
11. SDS-PAGE Electrophoresis
Separate the eluted proteins using SDS-PAGE.
12. Western Blot Analysis
Transfer the proteins from the gel onto a membrane, and detect the target protein or its SUMOylated form using specific antibodies via immunoblotting.
When performing endogenous SUMO Co-IP, it is essential to use optimized buffer systems and experimental conditions that preserve SUMO modifications and prevent protein degradation. Additionally, proper optimization of experimental parameters and inclusion of appropriate controls are critical for ensuring data reliability and interpretability.
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