Protein Crystals Cryo Protecting Guidelines
Preparation of cryo protecting solution
The most popular cryo protectants are: Glycerol MPD, PEG400 PEG1K, PEG MME 550 Ethylene glycol Glucose
Prepare set of solutions with increasing concentration of cryo agent say, from 15% to 35-40% with 5% step (solution should contain, of course, the main precipitant and buffer as in crystallization conditions). It is good idea to increase concentration of main precipitant by 0.1-0.2M since most cryo protecting agents can increase solubility of protein, for example, glycerol.
Testing for cryo conditions - which concentration of cryo is good
Dip the clean empty cryo loop into solution of cryo and then quickly (as quick as possible) mount under liquid nitrogen stream. The loop with cryo should be clear and transparent. Take the image as usual like the crystal is in the loop. Test solutions with increasing concentration of cryo until the good cryo protecting conditions are found.
The image with good cryo protecting concentration should not contain ice rings, only the diffuse band of diffraction.
Testing the protein crystal for good cryo protection
The simple procedure of cryoprotecting is to place protein crystal from the crystallization drop to the drop of solution with good cryo protecting concentration, soak crystal for 15-30 seconds and then quickly mount crystal under stream of liquid nitrogen. If the crystal did not tolerate this procedure, the idea is to gradually increase concentration of cryo (usually in steps of 5%) and soak protein crystal in the set of cryo solutions, until the good cryo protecting concentration is reached. Sometimes increasing the soaking time at each step can also help.