Superdex 200 hr 16 60

The SKP1-SKP2 complex and p27 were purified by affinity chromatography on a glutathione-sepharose column (Merck) equilibrated in mHBS as described previously^{46 (link)}. Proteins were eluted by 20 mM glutathione in mHBS pH 8.0, cleaved with 3C protease (1:50 w/w) and further purified using size exclusion chromatography (SEC) (Superdex 200 HR 16/60 (Cytiva)) equilibrated in 20 mM Tris pH 7.8, 300 mM NaCl, 0.5 mM TCEP. T160pCDK2-cyclin A complex was prepared as previously described^{56 (link)}. Briefly, a glutathione-sepharose column equilibrated in mHBS was charged with GST-T160pCDK2, washed to baseline and then used as an affinity column to purify untagged cyclin A. After washing to baseline T160pCDK2-cyclin A was eluted with 20 mM glutathione in mHBS, cleaved with 3C protease and subjected to SEC (Superdex 200 HR 16/60 (Cytiva)) equilibrated in 20 mM Tris pH 7.8, 300 mM NaCl, 0.5 mM TCEP. Co-eluting GST dimer was removed through subtractive GST. CKS1 was purified by affinity chromatography on an HiTrap His column (Cytiva) equilibrated in 40 mM HEPES pH 7.0, 200 mM NaCl, 25 mM imidazole and eluted with a gradient of 300 mM imidazole before loading onto a Superdex75 column (Cytiva) equilibrated in 20 mM Tris pH 7.8, 300 mM NaCl, 0.5 mM TCEP. The His6 tag was not cleaved.