Table 1.

Troubleshooting table

Step	Problem	Possible reasons	Possible solutions
Section 2.2 Step 2	The amount of total protein cell lysates using SDS lysis- based protein extraction method is low	Large volume of SDS buffer was used in Section 2.1. Small volume of SDS buffer was used in Section 2.1 Inaccurate Bradford assay	To avoid loss of protein, extracts should not be too dilute. If a high volume of lysis buffer was used to avoid sample viscosity, try DNase digestion or sonication for genomic DNA fragmentation. Using insufficient volume of lysis buffer prevents cell lysis. 40ul of final volume per 5×105 cells is recommended. SDS could interfere with Bradford assay. Alternative protein assays, such as Lowry protein assay, may be required for accurate protein quantification.
Section 2.2 Step 7	The SUMO-modified form of Daxx is not detected by Western blotting	Western blot exposure too short Modified and unmodified forms not sufficiently separated	Because only a small fraction of endogenous Daxx is sumoylated, at least 50ug cell lysates are required for Western blot analysis. Make sure blots are exposed overnight in order to detect SUMO modification. The unmodified form of Daxx is detected as an approximately 120kDa band by SDS-PAGE while the SUMO-modified Daxx is detected as ~140kDa. Run6% or 7.5% PAGE gel for 4–5 hours (or longer if necessary) at 60V to nicely separate the two Daxx species. This will place unmodified Daxx very near the bottom of the gel.
Section 2.3 Step 3	Unequal protein expression levels	Used CMV promoter-driven plasmids	pp71 transactivates the CMV promoter. Co- expression of pp71 would increase the expression of other co-transfected plasmids that are under control of CMV promoter. Use other promoters (e.g., SV40 promoter).
Section 2.4	IP using SUMO-1 antibody did not show Daxx by Western blotting	Low antibody binding efficiency Lost the sumoylated Daxx during IP	Use antibody up to ~20ug/IP reaction. Small IP volume (~500ul) is favorable. Incubate antibody overnight (>12 hours) at 4°C. Because IP is performed in RIPA condition (non- denaturing condition), SUMO proteases may be partially active even in the presence of NEM and IAA, or become active during long IP procedure. Crosslinking may be helpful to solve the problem if this is the case.
Section 2.4	Difficult detection of endogenous protein sumoylation	Low sumoylation due to combination of low levels of free pool of SUMO and substrate protein in cells	Use cells that constitutively express SUMO proteins or SUMO-Ubc9 fusion proteins. Despite its weakness of artificial system, increased SUMO machinery can be a useful way to detect endogenous substrate sumoylation.