GST Pulldown Assay for Protein Interactions

GST pulldown assays were performed as previously described (Shestakova et al., 2010 (link), Adell et al., 2014 (link)). Pierce magnetic glutathione beads (Thermo, 78602) were incubated in 0.1% BSA in ATPase buffer overnight. 5μg of GST-tagged proteins were bound to beads for 2 hours at 4°C, washed and optionally incubated with 500 ng of Cmp7-3xFLAG and/or Vps32-3xMyc for 1 hour at 4°C, followed by 5 five washing steps in ATPase buffer (+/- ATP) with 600mM NaCl. 500 ng of Vps4^E233Q -3xHA or Vps4-3xHA was added in the presence or absence of 1mM ATP for 10 minutes at room temperature. After three washing steps in ATPase buffer with 300mM NaCl, proteins were eluted from beads using sample buffer (2% SDS, 100mM Tris 6.8, 10% glycerol, 5% beta-mercaptoethanol, 0.01% bromophenol blue) at 96°C for 10 minutes. Samples were separated on a 12.5% SDS Page. Proteins were either stained by Brilliant Blue Coomassie or subjected to Western blotting.