Cryoprotection and Freezing of Protein Crystals
Purpose:
To prevent radiation damage to protein crystals during data collection crystals are frozen and maintained at -173o C under a stream of Nitrogen. To prevent ice crystals from forming during the process of freezing it is necessary to soak crystals in a series of solutions with increasing cryoprotectant concentration. Good cryoprotectants are small molecules such as:glycerol, PEG-400, methyl-pentanediol (MPD), and sucrose. The appropriate cryoprotectant to use, amount of soak time, final cryoprotectant concentration, and the rate at which the cryoprotectant is increased must be determined by the user, exposing a frozen loop containing the final cryoprotectant solution but no crystal.
Cryoprotecting Protocol:
1.

2.

 Remove crystals from mother liquor and place in a sitting drop well filled with mother liquor containing the lowest concentration of cyroprotectant.

After appropriate time transfer crytals to wells filled with mother liquor containing the next higher concentration ofcryoprotectant. Repeat this step until crystals are soaking in final concentration of cryoprotectant.

Alternatively, except for the solution immediately around the crystal, simply empty the well and fill it with the next solution with higher cryoprotectant concentration. This prevents transferring delicate crystals from one well to another.
Freezing Protocol: (using Hampton cryoloops)
1. 2. 3. 4. When ready, fill up cryovial with liquid propane.
Scoop up a single crystal in a cryoloop mounted on appropriate size cryoloop pin. Quickly plunge cryoloop into cryovial and screw in place.
Plunge cryovial into liquid Nitrogen. Wait for 10 to 15 minutes for propane to freeze. For long term storage place in liquid Nitrogen storage dewar.