1 Concentrate protein samples using Centricon Concentrators 5K. From this point on the work is conducted with concentrated proteins and extra care should be taken. The Centricon concentrators must be autoclaved and disposed of as indicated for autoclaved material in this document.
2 Exchange buffer to Crystallization Buffer:
• 20 mM HEPES pH 8.0, 250 mM NaCl, 2 mM DTT
3 Depending on protein solubility, concentrate to 500 uL to 1 mL (from this point forward the protein samples should be stored in screw cap microfuge tubes (e.g. Fisher # NC9205820) and transported in a closed secondary container such as a Rubbermaid sealed top container).
4 Measure protein concentration
5 You are now ready to set up crystallization experiments
6 Make sure to use the Mosquito with enclosure for experiments using unknown proteins of RG2 microbes.
7 The Mosquito surfaces and box enclosure must be treated with 10% bleach after crystallization setup. The box enclosure must be wiped down on a weekly basis and must be indicated on the attached sin-up sheet. This will ensure compliance.
8 Transfer concentrated proteins in secondary container with closed lid. In case of spills please refer to the spill cleanup procedure.
9 The crystallization plates must be clearly labeled containing the unknown targets of RG2 microbes.