How to perform endogenous Co-IP with SUMO?
The endogenous Co-Immunoprecipitation (Co-IP) experiment for SUMO (Small Ubiquitin-like Modifier) typically follows these steps:
1. Cell Lysis:
First, cells need to be collected. For SUMOylation studies, transiently transfected cells or cells treated under specific conditions to increase the level of SUMOylated proteins are typically used.
2. Preparation of Lysis Buffer:
Use a lysis buffer containing protease inhibitors and SUMOylation inhibitors such as N-Ethylmaleimide (NEM). NEM is used to inhibit deSUMOylating enzymes, preserving the SUMOylation modifications.
3. Cell Lysis:
Lyse the collected cells in the lysis buffer. This step is typically performed at low temperatures to prevent protein degradation and deSUMOylation.
4. Centrifugation:
Centrifuge the lysed cell supernatant to remove cell debris.
5. Pre-clearing:
Pre-clear the supernatant to remove non-specific binding proteins, usually using A/G beads or corresponding control antibodies.
6. Add Antibody:
Add a specific antibody against the target protein to the pre-cleared supernatant to bind the SUMOylated proteins. Sometimes, an antibody directly against SUMO can be used for immunoprecipitation.
7. Incubation:
Incubate the mixture overnight at low temperatures to promote binding of the antibody to the target protein.
8. Capture Immune Complex:
Add protein A/G beads to capture the antibody-protein complex.
9. Washing:
Wash multiple times to remove non-specifically bound proteins.
10. Elution:
Elute the immune complex using SDS-PAGE loading buffer.
11. Protein Electrophoresis:
Perform electrophoresis on the eluted samples using SDS-PAGE.
12. Protein Blotting (Western Blot):
Transfer the electrophoresed proteins onto a membrane and use specific antibodies to detect the target protein or its SUMOylated form.
When conducting a SUMO endogenous Co-IP experiment, it is important to ensure the use of appropriate buffers and conditions to protect the SUMOylation state and reduce protein degradation and deSUMOylation. Additionally, optimization of the experiment and control experiments are also crucial steps.
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