CryoPro

Applications

  • Water soluble cryoprotectant reagent set designed for the cryopreservation of biological macromolecular crystals

Features

  • 24 Cryoprotectants
  • 1.5 mL volume
  • Water soluble compounds
  • Formulated using Type 1+ ultrapure water, 18.2 MΩ•cm resistivity at 25°C, <5 ppb Total Organic Carbon, <1 Bacteria (CFU/mL), <0.03 Endotoxin (EU/mL)
  • Organic, non-volatile, Osmolyte, Polyol, Polymer/Polyol, Polymer, Solvent, Sugar, and Salt

Description

Just as with identifying and optimizing reagents for crystallization, the identification and optimization of a suitable cryoprotectant involves some trial and error as well as screening. A suitable cryoprotectant, when mixed with the crystal and crystallization reagent will cool to cryogenic temperature without ice formation and not damage 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 loop a small amount of this mixture using a CryoLoop. Next, the CryoLoop containing the mixture is cooled in a bath of liquid nitrogen or in a cryostream. One then inspects for ice formation either visually under a microscope or with X-ray diffraction. Upon cooling, a transparent drop and X-ray diffraction pattern mostly free of powder diffraction rings or “ice rings” indicates success where the appearance of a cloudy drop or “ice rings” indicates an inappropriate cryoprotectant concentration or cryoprotectant. Incrementally increase the concentration and/or alter composition of the cryoprotectant serially, 5 to 10% and repeat the procedure until the drop remains clear when cooled. Once a cooled, clear drop is achieved, this is typically a good starting point for cryopreservation of the crystal. Next, one needs to test the stability as well as the X-ray diffraction of the crystal in the cryoprotectant. It is not essential that these preliminary diagnostic tests be performed, but they can provide useful data towards identifying an appropriate cryoprotectant and concentration for your crystal. Typically, the addition of 10 to 30% cryoprotectant to the mother liquor will be sufficient as a reasonable starting point.

Some crystals can be dipped or washed quickly (2 seconds) in a simple cryoprotectant such as 30% Glycerol for successful cryopreservation. But, when this fails, a rational assay of each cryoprotectant with incremental increases or decreases 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.



CAT NO

HR2-074

NAME

CryoPro

DESCRIPTION

24 x 1.5 mL, tube format

PRICE

$362.00

cart quote

Support Material(s)

Related Item(S)

References

1. Boutron, P. (1987). Non-equilibrium formation of ice in aqueous solutions: efficiency of polyalcohol solutions for vitrification. In: Pegg, D.E. & Karow, A.M. Jr. (eds). The biophysics of organ prese

2. Garman, E.F., & Mitchell, E.P. (1996). Glycerol concentrations required for cryoprotection of 50 typical protein crystallization conditions. J. Appl. Cryst. 29, 584-587.

3. Garman, E.F., & Schneider, T.R. (1997). Macromolecular Cryocrystallography. J. Appl. Cryst. 30, 211-237.

4. Hope, H. (1988). Cryocrystallography of biological macromolecules: a generally applicable method. Acta Cryst. B 44, 22-26.

5. Kottke, T., & Stalke, D. (1993). Crystal handling at low temperatures. J. Appl. Cryst. 26, 615-619.

6. Kwong, P.D. Liu, Y. (1999). Use of cryoprotectants in combination with immiscible oils for flash cooling macromolecular crystals. J. Appl. Cryst. 32, 102-105.

7. Mehl, P. (1989). Experimental dissection of devitrification in aqueous solutions in 1,3-butanediol. Cryobiology. 26, 567-568.

8. Parkin, S., & Hope, H. (1998). Macromolecular cryocrystallography: Cooling, mounting, storage and transportation of crystals. J. Appl. Cryst. 31, 945-953.

9. Petcock, J.M., Wang, Y.-F., DuBois, G.C., Harrison, R.W., & Weber, I.T. (2001). Effects of different post-crystallization soaking conditions on the diffraction of Mtcp1 crystals. Acta Cryst. D57, 763-

10. Petsko, G.A. (1975). J. Mol. Biol. 96, 381-392.

11. Rodgers, D.W. (1994). Cryocrystallography. Structure. 2, 1135-1140.

12. Schneider, T.R. (1997). Cryocrystallography of biological macromolecules. Acta Physica Polonica A. 91, 739-744.

13. Teng, T.-Y. (1990). J. Appl. Cryst. 23, 387-391.

14. Walker, L.J., Moreno, P.O., Hope, H. (1998). Cryocrystallography: effect of cooling medium on sample cooling rate. J. Appl. Cryst. 31, 954-965.

15. Watenpaugh, K.D. (1991). Curr. Op. Struct. Biol. 1, 1012.

16. Proline: Mother Nature\\\\\\\\\\\\\\\'s cryoprotectant applied to protein crystallography. Pemberton TA, Still BR, Christensen EM, Singh H, Srivastava D, Tanner JJ. Acta Crystallogr D Biol Crystallogr. 2012 Aug;68(Pt 8):1010-8. doi: 10.1107/S0907444912019580. Epub 2012 Jul 17.

17. Flash freezing of protein crystals: investigation of mosaic spread and diffraction limit with variation of cryoprotectant concentration E. P. Mitchell and E. F. Garman. J. Appl. Cryst. (1994). 27, 1070-1074 [ doi:10.1107/S0021889894008629 ]