Detecting Protein Sumoylation in Cells

Examination of protein sumoylation was carried out as previously described [8 (link)]. Briefly, cell lysates were prepared using bead beating methods under denaturing conditions to prevent desumoylation during protein extraction and minimize co-purification of associated proteins. Diluted protein extracts were then immunoprecipitated, using IgG-sepharose (Sigma) to pull down TAP-tagged Pol2, or Protein G-agarose plus anti-HA (12CA5) antibody to pull down HA-tagged Pol2. Immunoprecipitated proteins were washed and eluted with loading dye before separation on standard SDS-PAGE gels and immunoblotting with anti-SUMO [12 (link)] and tag-specific antibodies including Peroxidase Anti-Peroxidase (Sigma-Aldrich, P1291) and anti-HA (Sigma, 12CA5). As for most sumoylated proteins, the sumoylated form of Pol2 is of low abundance and not seen under normal exposure using anti-tag antibodies but can be readily detected by anti-SUMO antibody. Standard methods for detecting protein levels in crude cell extracts and protein interactions by co-IP were used. For experiments in Fig 2D, DNase was added to remove DNA before immunoprecipitation, and anti-Flag antibody was obtained from Sigma-Aldrich (F1804).