Cryo-Protectants and Freezing

Cryoprotectants include: Polyols, organics, oils, polymers, sugars, & salts

From Hampton Research:

As in selecting reagents for crystallization, the selection of a suitable cryoprotectant involves some trial and error as well as a screening.1-15 A suitable cryoprotectant, when mixed with the crystal and crystallization reagent, will cool to cryogenic temperature without ice formation and damage to the crystal. To assay for the proper concentration of cryoprotectant in the reagent used to grow the crystal, one can mix the cryoprotectant with the crystallization reagent and employ the desired cooling method (for example, place the solution in a CryoLoop™ and place the CryoLoop in a cryostream). Observe for ice formation either visually or with x-ray diffraction. Upon cooling, a transparent drop and x-ray diffraction pattern, free of powder diffraction rings or “ice rings” indicates success. The appearance of a cloudy drop or “ice rings” indicates an inappropriate cryoprotectant concentration or cryoprotectant. Incrementally increase the concentration and/or composition of the cryoprotectant serially 5 to 10% and repeat until the cooled drop remains clear while in the cryostream. Once a clear drop is achieved in the cryostream, this is typically a good starting point for cryopreservation of the crystal.

Some crystals can simply be dipped or washed quickly in a simple cryoprotectant such as 30% glycerol for successful cryopreservation. However, when all else fails, a rational assay of each cryoprotectant with incremental increases in cryoprotectant concentration as well as a test of mixtures (for example a mixture of sugars, or a sugar mixed with ethylene glycol) may be required to determine the best cryoprotectant for a crystal.

From: "Flash Cooling Protein Crystals: Estimate of Cryoprotectant Concentration Using Thermal Properties," Binal N. Shah, Unmesh Chinte, Stephen J. Tomanicek, B. Leif Hanson, and Constance A. Schall, Cryst. Growth Des., 2011, 11(5), pp 1493–1501. 


X-ray diffraction from protein crystals is routinely measured at cryogenic temperatures, primarily to minimize radiation damage. Protein crystals and the surrounding mother liquor have high water content, which can lead to ice formation when samples are cooled to cryogenic temperatures. Cryoprotectants are added to the aqueous mother liquor solutions to achieve both vitreous water and to retain protein crystal integrity. Finding a minimum cryoprotectant concentration to avoid ice formation is a trial and error procedure, and valuable crystals are often lost in this exercise.


The 3-dimensional structure of macromolecules is obtained largely from X-ray diffraction data measured on crystals at cryogenic temperatures.(1-4) Exposure of protein crystals to ionizing X-rays results in the formation of photoelectrons and free radicals. These in turn react with the protein, breaking disulfide and other bonds, disordering the lattice and reducing the crystal lifetime in the X-ray beam.(5) At cryogenic temperatures, the diffusion of these radicals is dramatically reduced, thus increasing the crystal lifetime.

Protein crystals contain 10 to 100 Å channels, filled with crystallizing or stabilizing solution. These solutions are mixtures of salts, precipitants, and buffer in an aqueous solution. On cooling to cryogenic temperatures, water within and around the crystal can form ice, giving rise to additional X-ray scattering from ice crystals and potentially disrupting the protein crystal lattice. Formation of crystalline ice can be avoided by rapidly cooling the protein crystal to form a vitreous (glassy) aqueous phase below the glass transition temperature. Pure water and many crystallization solutions require very high cooling rates to achieve this vitreous phase that generally are unavailable with common cryogens. The vitrification of aqueous solutions in protein crystals can be achieved by mixing cryoprotectants with the crystal stabilizing solution.(1-3, 6) Cryoprotectant addition decreases the melting point and the homogeneous nucleation temperature of water, decreasing the temperature window of ice formation.(7, 8) Commercial cryostat cooling rates are then rapid enough to form vitreous solutions. However, cryoprotectant selection and concentration is often a trial and error procedure, leading to losses of valuable protein crystals in the process.