Figure 4: A schematic overview of the PRISM double purification strategy.

(a) 1. Cells are lysed under denaturing conditions, inactivating endogenous proteases. 2. SUMOylated proteins are pre-enriched using, in this case, Ni-NTA pulldown to capture the histidine tag. 3. SUMOylated proteins are acetylated on-beads using SNHSA under highly denaturing conditions, enabling efficient blocking of lysines. Proteins are eluted using a buffer compatible with a second chemical labelling step. 4. Following elution, lysine-blocked SUMOylated proteins are treated with SENP2, efficiently freeing up the lysines SUMO-2 was conjugated to. 5. Freed lysines are biotinylated using SNHSSSB. 6. Biotinylated SUMO target proteins are enriched using avidin pulldown, and eluted sequentially using DTT and LDS elution buffers. Finally, SUMO target proteins may be analysed by various biochemical methods, such as immunoblotting. (b) SUMOylated proteins were purified from HeLa cells expressing lysine-deficient His-tagged SUMO-2 (PD: His), using PRISM as described in a. For diagnostic reasons, the assay was performed with and without the use of SNHSA to block lysines. Samples were analysed by immunoblotting for the presence of TRIM33. Non-blocked TRIM33 eluted after Ni-NTA pulldown, Step 2, is indicated. Lysine-blocked TRIM33 eluted after Ni-NTA pulldown, Step 3, is indicated. The experiment was performed in biological duplicate. (c) As in b, but using a SUMO-2/3 antibody. (d) As in b, but additionally eluted proteins were either treated with SENP2 or mock treated. Lysine-blocked TRIM33 eluted after Ni-NTA pulldown, Step 3, is indicated. Lysine-blocked TRIM33 that was successfully deSUMOylated by SENP2, Step 4, is indicated. The experiment was performed in biological duplicate. (e) As in d, but using a SUMO-2/3 antibody. (f) All samples described in d were treated with SNHSSSB, and enriched using avidin pulldown (PD: Biotin). Elution was initially performed with DTT, and second with LDS. Samples were analysed by immunoblotting for the presence of TRIM33. On the long exposure, TRIM33 that was lysine-blocked, successfully deSUMOylated by SENP2 and specifically biotinylated and purified, Step 6, is indicated. On the short exposure, multi-biotinylated TRIM33 is indicated. The experiment was performed in biological duplicate. (g) Same as in f, but using a SUMO-2/3 antibody.