Protein sumoylation is an important post-translational modification process involving the covalent binding of small ubiquitin-like modified proteins (SUMOs) to target proteins. Sumoization plays an important role in many biological processes, such as nuclear transport, transcriptional regulation, cell cycle progression, and stress response. The following are commonly used methods to detect protein sumoization:
1.western blot：
Using specific anti-SUMO antibodies, proteins can be directly detected if they have been SUMO-modified. Sumoization often results in an increase in the molecular weight of the protein, which can be observed after electrophoretic separation.
2.Immunoprecipitation (IP) + Western blot:
The protein of interest is first precipitated from the cell lysate with a specific antibody, and then the SUMO status is detected by Western blot using an anti-SUMO antibody.
3.Mass spectrometry analysis after affinity purification:
With affinity purification of the SUMO binding site, SUMO-ylated proteins can be enriched from complex samples and then identified using mass spectrometry.
4.Bioluminescence Resonance Energy Transfer (BRET):
Intracellular detection of the dynamic process of sumoization. It typically involves attaching fluorescent protein and fluorescent protein tags to the target protein and SUMO, respectively, and then detecting their interactions by measuring the energy transfer between the two.
5.Confocal Fluorescence Microscopy:
Using fluorescently labeled SUMO and the protein of interest, the location and dynamics of SUMO-based modifications can be visualized directly within the cell.
6.Enzyme-linked immunosorbent assay (ELISA):
Using specific anti-SUMO antibodies, it is possible to quantify SUMO-ated proteins.