Laboratory name
Washington University
Default Washington University crystallization setup: Purified proteins are exchanged into crystallization buffer (20 mM HEPES pH 7.4, 25 mM NaCl) using a separate Amicon Ultra centrifugal filtration device of the appropriate MWCO for each protein. The centrifugation is continued until the final protein concentration reaches between 10-20 mg/ml. Place 2ul of the sample into the NanoVue spectrophotometer (GE-Healthcare) to obtain an OD. The approximate concentration can obtained for each protein from its OD using the protein’s theoretical extinction coefficient calculated by ProtParam (Expasy website). The concentration process is monitored closely for signs of precipitation to prevent exceeding sample solubility. Depending on the particular protein, 2mM DDT may be added to inhibit protein cross-linking by disulfide bond formation between surface-exposed cysteine. Crystallization trials are carried out in 96 well “MRC 2” plates (Hampton Research Cat# HR3-083) by sitting-drop vapor-diffusion technique. Each well is filled with 50 ul of commercially available 96 well crystallization screens (JCSG, PEGs, Index, Cryo, et cetera) by hand using a multichannel pipette. The crystallization drops are setup using a Mosquito nano-liter high throughput liquid handling robot (TTP labtech). Typically 0.3 ul protein and 0.3 ul well solution are combined for each drop. The plates are sealed with UV-transparent ClearSeal Film (Hampton Research Cat# HR4-521). Each plate must contain a 4 digit WU-ID number, the date, the experimenter’s name, the protein’s name, the screen name, and type of protein (native or SeMet). The information for each plate must be entered into CrystalTrak software, a barcode printed, and an appropriate observation schedule chosen before placing the plate into the Gallery 700 side of the Minstrel-HT (Rigaku). Monitor the drops for crystal growth and optimize in hanging drop Linbro plates using the Alchemist HT screen maker (Rigaku). All crystallization experiments are performed at 20 C. Protein samples can be stored for short term at 4C, but for longer term storage proteins should be flash frozen in aliquots and kept at -80C.