Hello! I'm looking for a protocol to extract proteins just to analyze SUMOylation by Western Blott. I don't want to analyze the SUMOylation of a target protein, just degree of sumoylation in different extracts.
Hi! Since SUMOylation is a covalent modification, I think you can use harsh lysis conditions such as RIPA buffer + sonication. In this way you should extract everything. You can even give a look to the following paper, they talk about ubiquitination assay in cellulo. If you want, instead recombinant Ubiquitin, put recombinant SUMO
If you are not interested in particular target proteins, then it may be more quantitative if you do a dot blot. Instead of running a gel and transfer, you simply spot the lysate directly on nitrocellulose membrane, then follow the usual block-primary-wash-secondary-wash-image protocol for an anti-SUMO antibody. Although, this will give signal not only for total SUMOylated protein, but also free SUMO...
thanks so much Beverley!! I was looking for a protocol and i read about a buffer 1% SDS or 8M urea...I'm not sure about what to do !! Just I want to see total sumoylation in different cell lines