I have conducted several western blot experiments to detect the SUMO-conjugates bands (or smear) in tobacco total proteins extract expressing the SUMO. But, I could not detect the SUMO-conjugated bands (see one of my WB results attached here). I have also used NEM inhibitor in the extraction buffer to block the activity of SUMO-protease proteins, but still unable to detect the SUMO-conjugate bands.

Is anyone has any experience on it? Why I could not detect the SUMO-conjugates profile in the plant total protein extracts (TPE)? Are there specific factors that requires special attentions when studying SUMO modification? Do I miss something in my WB experiments? Or do I need to first enrich the SUMO-conjugate proteins from TPE using beads and then do the WB?

Any suggestions or recommendations would be greatly appreciated.

Thank you!