Both cover slips and 6-well plates were precoated with poly-D-lysine

e from Life Technologies. Protein purification FLAG-Parkin. Frozen pellets were resuspended in buffer A and lysed using a microfluidizer. The lysate was cleared and the supernatant agitated gently with Anti-FLAG M2 agarose for 1 hr at 4uC. The beads were washed with 10 column volumes of buffer A and the Parkin protein was eluted with 10 column volumes of buffer A containing 100 mg/ml 3X FLAG peptide. After elution, 1mM TCEP was added and the protein was diluted with 50 mM Tris to reduce the salt concentration to 20 mM NaCl. The protein was then loaded onto a Mono Q HR 10/10 anion exchange column that had been pre-equilibrated in buffer B. The column was developed with a gradient of 02500 mM NaCl over 34 column volumes, and the protein was eluted at 100140 mM NaCl. Collected fractions were then concentrated and injected onto a HiLoad 26/60 Superdex 200 column that had been preequilibrated in buffer C. The column was eluted with 1.5 CV of buffer 2 Parkin SPR Fragment Screening C. The concentration of FLAG-Parkin was established using the calculated extinction coefficient of 63,440 cm21 M21. R0RBR. Frozen pellets were resuspended 15930314 in buffer A and lysed using a microfluidizer. The lysate was cleared and the supernatant agitated gently with high performance Ni sepharose for 1 hr at 4uC. The beads were washed with 10 column volumes of buffer A containing 20 mM imidazole and then washed with 10 column volumes of buffer A containing 40 mM imidazole. The R0RBR protein was eluted with 10 column volumes of buffer A containing 200 mM imidazole. After elution, the protein was dialyzed into 50 mM Tris for 2 hr at 4 uC to reduce the salt concentration. The protein was then loaded onto a Mono Q HR 10/10 anion exchange column that had been pre-equilibrated in buffer B. The column was developed with a gradient of 02500 mM NaCl over 50 column volumes and the protein was eluted at 1132180 mM NaCl. The Sumo tag was then removed by incubation with SENP1 for 2 hr at 4uC. Following the incubation, 10 mM imidazole was added to the cleavage reaction and the reaction was DMXB-A custom synthesis purified over a high performance Ni sepharose column to remove the Sumo tag and the SENP protease. The Ni column was washed with 10 CV of buffer A. Both the wash and the flow thru from the Ni column were collected and injected onto a HiLoad 26/60 Superdex 200 column that had been preequilibrated in buffer C. The concentration of R0RBR was established using the calculated extinction coefficient of 46,940 cm21 M21. Ubl and 26506265 Ubch7. Frozen pellets were resuspended in buffer A and lysed using a microfluidizer. The soluble fraction was collected after centrifugation at 45,000 g for 30 minutes and purified over nickel sepharose using batch mode 1 hour binding at 4C. The beads were washed with 10 column volumes of buffer A containing 20 mM imidazole and followed by 10 column volumes buffer A containing 40 mM imidazole followed by elution of the proteins with 8 column volumes of buffer A containing 300 mM imidazole. A small fraction of the nickel purified proteins were dialyzed against 50 mM Hepes, pH 8.8, 0.05% Tween-20, 0.01% pluronic F127 and 10% glycerol for Biacore experiments. The concentrations of UBCH7 and UBL were established using the calculated extinction coefficient of 18450 cm21 M21 and 11000 cm21 M21, respectively. To remove the N-terminal 8xHis tag from Ubch7, the remaining protein was incubated with TEV overnight at 4uC via dialysis in buffer A. The cleaved material was purified over a high