I am currently conducting a Western blot analysis on A. evenia and tobacco (Nb) plant total protein extracts expressing SUMO protein in order to detect SUMOylation using an anti-SUMO antibody. However, despite following standard protocols and utilizing NEM inhibitor (a SUMO protease inhibitor) in the extraction buffer to block the activity of SUMO protease proteins (in case of endogenous SUMO protease proteins), I have been unable to detect the expected SUMO-conjugated bands. Interestingly, I have come across published data showing a high molecular weight smear of SUMO-conjugated bands in similar experiments (see attached image).
I am reaching out to seek advice from the community. Are there any crucial steps in the SUMO modification process that may have overlooked? Additionally, are there specific factors that require special attention when studying SUMO modification in plant systems? Do I miss something in my WB experiments?
Any insights, recommendations, or troubleshooting tips would be greatly appreciated.
Thank you!