The protein was diluted to a concentration of 8 mg/ml by adding 10 mM Tris/HCl, pH 8.5, concentrated DTT, and concentrated NaN3 so that the final buffer consisted of 10 mM Tris/HCl, pH 8.5, 10 mM DTT and 3 mM NaN3.
Protein was eluted from the beads twice with 20 mM Glutathione pH 8, concentrated on Vivaspin 500 columns (Sartorius) and dialysed against 30 mM Hepes pH 7.4, 5 mM MgCl2, 100 mM KCl, 10% Glycerol, 0.1 mM AEBSF before use in kinase assays.
The elute containing the purified enzyme was immediately desalted with 50 mM Tris HCl buffer (pH 7.5), concentrated by centrifugation at 4 °C (Amicon Ultra-15, Merck Millipore), and immediately used for activity assays.
The superhydrophobicity is retained even in highly corrosive acidic and basic conditions (pH 1.29 13.54, concentrated HNO3 exposure and ammonium hydroxide solution), as well in a wide humidity range (35 83%).
Fractions containing the desired protein were dialyzed against Hepes buffer (50 mM, pH 7.0), concentrated to 5 mg/ml.
The fractions containing PncA were eluted by a linear gradient of 0 to 1M NaCl in Bis-Tris 40 mM pH 6.0, concentrated with Amicon ultra 15 ml (Millipore) and loaded onto a gel filtration Superdex 75 (Pharmacia Co. Ltd., Sweden) previously equilibrated with Bis-Tris 40 mM pH 6.0.
The resin was centrifuged for 2 min at 2000× g and washed three times at RT with 10 ml washing solution containing 100 mM sodium phosphate buffer pH 8. Concentrated milk and brain PrPC was denaturated in SDS sample buffer and heated at 70°C for 10 min and at 95°C for 5 min, respectively.
For co-crystallization, Uba2ufd was purified by gel filtration chromatography using a SD75 column (GE) in 25 mM Tris-HCl, 0.15 M NaCl, 5 mM DTT, pH 7.6, concentrated to 22 mg/ml (Bio-Rad Protein Assay), aliquotted, flash-frozen in liquid nitrogen, and stored at −80°C until further use.
Ubc9 was purified to homogeneity by cation exchange with a homemade column packed with ResourceS resin (GE) using a NaCl gradient in 20 mM HEPES, 1 mM DTT, pH 7.0, concentrated to 27 mg/ml, aliquotted, flash-frozen in liquid nitrogen, and stored at −80°C until further use.
Trophonts, tomonts and theronts were washed with PBS (pH 7.4), concentrated with a centrifuge (Beckman Coulter, Inc., Miami, FL) at 228 × g for 5 min and discarded supernatant.
