High throughput crystallization screen kits

Initial crystallization screens were performed using a Phoenix crystallization robot (Art Robbins Instruments) and high-throughput crystallization screen kits (Hampton Research, Qiagen, or Emerald BioSystems), followed by extensive manual optimization. The best single crystals were grown at 18 °C by the hanging-drop vapor-diffusion method in a 1:1 (v/v) ratio of protein and reservoir, as follows. (1) 4H11-scFv was crystallized with a reservoir solution composed of 0.1 M sodium citrate tribasic dihydrate (pH 5.0) and 20% polyethylene glycol (PEG) 4 K. Micro-seeding was necessary to obtain single crystals. (2) 4H11-scFv-MUC16-target complex was crystallized using a reservoir of 0.1 M sodium citrate tribasic dihydrate (pH 5.0), 10 mM barium chloride dihydrate, and 27% methoxypolyethylene glycol 5000 (PEG MME 5 K).

Initial crystallization screens were performed using a Gryphon crystallization robot (Art Robbins Instruments, Mountain View, CA, USA) and high-throughput crystallization screen kits (Hampton Research and Qiagen). Extensive manual optimizations were performed at 18 °C. The best single crystals of LCH_N/E1–JLE-E5 were grown by the sitting-drop vapor diffusion method at a protein concentration of 5 mg/mL with a reservoir solution containing 0.15 M Ammonium sulfate, 14% PEG 4000, 0.1 M Hepes (pH 7.0) when proteins were mixed with reservoir solution at 1:1 (v/v) ratio. Crystals of LCH_N/E1–JLE-E9 were obtained by the sitting-drop vapor diffusion method at a protein concentration of 6 mg/mL with a reservoir solution containing 12% PEG 20K, 0.1 M sodium citrate (pH 5.8) when proteins were mixed with reservoir solution at 2:1 (v/v) ratio.

Initial crystallization screens were performed using a Gryphon crystallization robot (Art Robbins Instruments) and high-throughput crystallization screen kits (Hampton Research and Qiagen). Extensive manual optimizations were performed at 18°C when proteins were mixed with reservoir solution at 1:1 ratio.