Ionic liquids have been found effective as additives in protein crystallization, with different ionic liquids used to increase crystallization rates and crystal size.^1-4 The inclusion of ionic liquids in crystallization experiments has been reported to lead to less crystal polymorphism as well as less precipitation at higher precipitant concentrations.²⁵ Ionic liquids have been used as additives to produce crystals in reagents that had previously not resulted in crystallization and results suggest ionic liquids may be applicable for the solubilization and crystallization of membrane proteins.²

Ionic liquids are organic salts with melting points below 100°Celsius. They are thermally stable, nonflammable and demonstrate very low vapor pressure. Ionic liquids are soluble in a variety of organic and inorganic reagents and can be highly water soluble. Ionic liquids can demonstrate a degree of localized structuring about each ion compared to materials composed of disassociated ions, setting them apart from salt solutions.^5-6 Ionic liquids can participate in ionic, hydrophobic and hydrogen bond interactions. Ionic liquids are often chaotropic, composed of low symmetry ions with charge delocalization and weak intermolecular interactions.¹ These organic salts generally consist of combinations of organic cations and either an organic or inorganic anion. Ionic liquids have been demonstrated to suppress protein aggregation and significantly increase protein folding yields.^7-8 Ionic liquids have been reported to stabilize protein activity and structure.^9-11 The inclusion of the ionic liquid 1-n-Butyl-3-methylimidazolium tetrafluoroborate (included in this kit) improved the thermal stability and solubility of integral membrane proteins for membrane proteomics study.¹²

Some ionic liquids, such as ethylammonium nitrate have water-like characteristics, including the capacity for hydrogen bonding and the promotion of micelle formation by some surfactants.¹³ Many ionic liquids are also organics acids and have ionic character in addition to the hydrophobic behavior, which makes them unique and useful solvents in protein chemistry.

Variation of the anion and the cation as well as the utilization of both soft (formate and acetate) and hard anions (nitrate) in the Ionic Liquid Screen reagents provides an additional dimension for evaluating the effects on ionic liquids on the solubility and crystallization of proteins. The Ionic Liquid Screen contains a set of 24 water soluble ionic liquids that comprise different cation (imidazolium, phosphonium, ammonium) and anion (borate, halides, sulfates, acetates, sulfonates, nitrates) structures for a diverse ionic liquid additive screen for use in improving the crystallization behavior and X-ray diffraction resolution of proteins.

Each of the 24 ionic liquids are preformulated in deionized water and sterile filtered using a 0.2 micron filter. Ionic liquid Screen contains 24 unique reagents, 0.5 milliliter each.

Ionic Liquid Screen

1. Pusey, M.L., Paley, M.S., Turner, M.B., and Rogers, R.D. 2007. Protein crystallization using room temperature ionic liquids. Crystal Growth & Design. 74:787-793.

2. Hekmat, D., Hebel, D., Sebastian, J. Schmidt, M., and Weuster-Botz, D. 2007. Advanced protein crystallization using water-soluble ionic liquids as crystallization additives. Biotech. Lett., 29:703-1711.

3. Garlitz, J.A., Summers, C.A., Flowers, R.A. and Borgstahl, G.E.O. 1999. Ethylammonium nitrate: A protein crystallization reagent. Acta Cryst. D53:2037-2038.

4. Judge, R.A., Takahashi, S., Longnecker, K.L. Fry, E.H., Abad-Zapatero, C., and Chi, M.L. 2009. The effects of ionic liquids on protein crystallization and X-ray diffraction resolution. Crystal Growth & Design 9:3463-3469.

5. Bowran, D.T., Hardacre, C. Holbrey, J.D., McMath, J.E., and Soper, A.K. 2003. J. Chem. Phys. 118:173-178.

6. Cadena, C., Zhao, Q., Snurr, and R.Q., Maginn, E.J. 2006. J. Chem. Phys. B 110:2821-2832.

7. Summers, C.A. and Flowers, R.A. 2000. Protein renaturation by the liquid organic salt ethylammonium nitrate. Protein Science 9:2001-2008.

8. Lange, C., Patil, G., and Rudolph, R. 2005. Ionic liquids as refolding additives: N′-alkyl and N′-(w-hydroxyalkyl) N-methylimidazolium chloride. Protein Science 14:2693-2701.

9. Lozano, P. , de Diego, T. Guegan, J.P., Vaultier, M., and Ibora, J.L. 2001. Stabilization of a-chymotrypsin by ionic liquids in transesterification reactions. Biotehnol. Bioeng. 75:363-369.

10. Baker, S.N., McCleskey, T.M., Pandy, S., and Baker, G.A. 2004. Fluorescence study of protein thermostability in ionic liquids. Chem. Commun. 2004:940-941.

11. De Diego, T., Lozano, P., Gmouth, S. Vaultier, M., and Iborra, J.L. 2004. Fluorescence and CD spectroscopic analysis of the a-chymotrypsin stabilization by the ionic liquid 1-ethyl-3-methyimidazolium bis[(trifluormethyl)sulfonyl]amide. Biotechnol. Bioeng. 88:916-924.

12. Sun, L., Tao, D., Han, B., Ma, J., Zhu, G., Liang, Z., Shan, Y., Zhang, L., and Zhang, Y. 2010. Ionic liquid 1-butyl-3-methyl imidazolium tetrafluoroborate for shotgun membrane proteomics. Anal. Bioanal. Chem. DOI 10.1007/s0216-010-4381-5.

13. Evans, D.F., Yamauchi, A., Roman, R., and Casassa, E.Z. 1982. J. Colloids Interface Sci. 88:89-96.

14. Proteins in Ionic Liquids: Current Status of Experiments and Simulations. Christian Schrode, Top Curr Chem (J). 2017; 375(2): 25.

15. Ionic Liquids as Stabilization and Refolding Additives and Solvents for Proteins. Fujita K. Adv Biochem Eng Biotechnol. 2018 Jul 13. doi: 10.1007/10_2018_65.

16. Fujita K. (2018) Ionic Liquids as Stabilization and Refolding Additives and Solvents for Proteins. In: . Advances in Biochemical Engineering/Biotechnology. Springer, Berlin, Heidelberg.

Hampton Research, first in crystallization since 1991, developing and delivering crystallization and optimization screens, reagents, plates, and other tools for the crystallization of biological macromolecules, including proteins (antibody), peptides (insulin), and nucleic acids (DNA).

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MembFac is based upon the highly effective biased sparse matrix methodology. The MembFac screen matrix is optimized for the crystallization of membrane proteins.

MembFac contains 48 unique reagent formulations. With this set of 48 conditions, numerous chemicals and chemical combinations are utilized, allowing one to evaluate a large variety of potential crystallization conditions. MembFac covers a broad pH range between 4 and 9.

The Rationale The basic rationale of MembFac is to perform a sparse matrix screen while changing the detergent dimension because detergents play an important factor in the crystallization of membrane proteins. A MembFac screen is to be completed for each detergent. Detergent screens are available separately.

The Method The membrane protein of interest is first isolated in the detergent which gives the highest stability and activity. The protein concentration should be approximately 10 to 20 mg/ml and the detergent concentration should be only slightly above the CMC. The sample is then set against MembFac, using all 48 solutions plus the screening detergent(s) of choice. The screening detergent concentration should be approximately one to three times the CMC. Hence, for each detergent screened, one would utilize approximately 1 to 2 mg of sample.

The MembFac protocol was used to crystallize membrane proteins including SQR, fumerate: quinone oxireductase, LHII, and LHCII.

Crystal Screen Lite is a sparse matrix of trial crystallization reagent conditions based upon the Crystal Screen™.³ The primary screen variables are salt, pH, and precipitant (salts, polymers, volatile organics, and non-volatile organics).² Crystal Screen Lite differs from the original Crystal Screen kit in that the primary precipitant reagents are one-half the concentration of that used in the original Crystal Screen formulation. The secondary salts, ions, and buffers remain at the original Crystal Screen concentration. Reducing the primary concentration of the primary precipitant results in a screen which is “more gentle” on the sample and typically produces much less precipitate conditions than the original Crystal Screen.

MembFac contains 48 unique reagents, 10 ml each.

MembFac HT contains 1 ml each of all 48 reagents from MembFac and reagents 1-48 from Crystal Screen Lite in a single Deep Well block format.

Ready-to-use reagents are sterile filtered and formulated with ultra-pure Type 1 water, using the highest purity salts, polymers, organics and buffers. Individual reagents are available through the Hampton Research Custom Shop.

MembFac

Crystal Screen Lite

MembFac HT

1. UCLA Crystallization Workshop, June 21, 1993.

2. Crystallization of nucleic acids and proteins, Edited by A. Ducruix and R. Giege, The Practical Approach Series, Oxford Univ. Press, 1992.

3. McPherson, A., Current approaches to macromolecular crystallization., Eur. J. Biochem. (1990) 189, 1-23.

4. Jancarik, J. and Kim, S.H., Sparse Matrix Sampling: a screening method for crystallization of proteins. (1991) J. Appl. Cryst., 24,409-411.

5. Protein and Nucleic Acid Crystallization. Methods, A Companion to Methods in Enzymology, Academic Press, Volume 1, Number 1, August 1990.

6. Structural and functional characterization of the N-terminal domain of the yeast Mg2+ channel Mrs2. M. B. Khan, G. Sponder, B. Sjoblom, S. Svidova, R. J. Schweyen, O. Carugo and K. Djinovic-Carugo. Acta Cryst. (2013). D69, 1653-1664.

Hampton Research, first in crystallization since 1991, developing and delivering crystallization and optimization screens, reagents, plates, and other tools for the crystallization of biological macromolecules, including proteins (antibody), peptides (insulin), and nucleic acids (DNA).

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By using sparse matrix sampling technology, The Natrix kits allow one to quickly test wide ranges of pH, salts, and precipitants using a very small sample of nucleic acid.

Natrix screens are unique in that rather than relying solely on the traditional nucleic acid precipitant MPD, Natrix screens also utilize Polyethylene glycols (PEGs) in a variety of molecular weights (200, 400, 4,000, 8,000) as well as 2-Propanol, Polyethylene glycol monomethyl ether (PEG MME), and 1,6-Hexanediol. Many of the polymeric and low molecular weight organic precipitants are combined with various monovalent salts as precipitating agents. This combination of salts and low molecular weight organics and polyalcohols, as well as the utilization of varying chain length PEGs, has proven to be a successful combination for producing nucleic acid and protein-nucleic acid complex crystals.

Natrix contains 48 unique reagents, 10 ml each and is based on the sparse matrix formulation first described by William Scott in 1995.

Natrix 2, an extension of Natrix, contains 48 unique reagents, 10 ml each. Natrix 2 is a biased sparse matrix screen based on extracting patterns from crystallization data as well as reagent formulations first described by Berger et al in 1996.

Natrix HT contains 1 ml of each reagent from Natrix and Natrix 2 in a single Deep Well block format.

Ready-to-use reagents are sterile filtered and formulated with ultra-pure Type 1 water, using the highest purity salts, polymers, organics and buffers. Individual reagents are available through the Hampton Research Custom Shop.

Natrix

Natrix 2

Natrix HT

1. Scott, W. G., Finch, J.T., Grenfell, R., Fogg, J., Smith, T., Gait, M.J., Klug, A., Journal of Molecular Biology (1995) 250: 327-332.

2. Purification, crystallization and preliminary X-ray analysis of the BseCI DNA methyltransferase from Bacillus stearothermophilus in complex with its cognate DNA. E. G. Kapetaniou, D. Kotsifaki, M. Providaki, M. Rina, V. Bouriotis and M. Kokkinidis. Acta Cryst. (2007). F63, 12-14.

3. Crystallization and preliminary X-ray diffraction analysis of an Escherichia coli tRNAGly acceptor-stem microhelix. C. Forster, M. Perbandt, A. B. E. Brauer, S. Brode, J. P. Furste, C. Betzel and V. A. Erdmann. Acta Cryst. (2007). F63, 46-48.

4. Combinatorial crystallization of an RNA-protein complex. Danielle Bodrero Hoggan, Jeffrey A. Chao, G. S. Prasad, C. David Stouta and James R. Williamson. Acta Cryst (2003). D59, 466-473.

5. Cocrystallizing natural RNA with its unnatural mirror image: biochemical and preliminary X-ray diffraction analysis of a 5S rRNA A-helix racemate. Volker A. Erdmanna et al. Acta Cryst. (2007). F63, 839-843.

6. Berger, et al., A Highly Effective 24 Condition Matrix for the Crystallization of Nucleic Acid Fragments. Acta Cryst. Section D. (1996) Vol. D52 Part 3, 465-468.

7. Crystallization and preliminary X-ray diffraction analysis of an Escherichia coli tRNAGly acceptor-stem microhelix. C. Förster, M. Perbandt, A. B. E. Brauer, S. Brode, J. P. Fürste, C. Betzel and V. A. Erdmann. Acta Cryst. (2007). F63, 46-48.

8. Adams, A., Nucleic Acids Research (2002) Vol. 30, 719-725.

9. Preliminary crystallographic analysis of mouse Elf3 C-terminal DNA-binding domain in complex with type II TGF-b receptor promoter DNA. Agarkar et al. Acta Cryst. (2009). F65, 1261-1263.

10. Crystallization and preliminary X-ray crystallographic studies of transglutaminase 2 in complex with Ca2+. T.-H. Jang and H. H. Park. Acta Cryst. (2014). F70, 513-516 [ doi:10.1107/S2053230X1400510X ]

Hampton Research, first in crystallization since 1991, developing and delivering crystallization and optimization screens, reagents, plates, and other tools for the crystallization of biological macromolecules, including proteins (antibody), peptides (insulin), and nucleic acids (DNA).

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The PCT kit contains 4 unique, preformulated, sterile filtered reagents used to evaluate protein concentration for crystallization screening. Initially, the sample protein is mixed with two of the reagents to determine if the protein concentration is appropriate for crystallization screening. If the protein is very sensitive to salt and polymer concentration, based on initial PCT results, the protein may be evaluated using a second set of PCT reagents. PCT results will then provide insight to either the appropriate sample concentration or indicate that other diagnostic testing such as native gel electrophoresis or dynamic light scattering should be performed to demonstrate sample homogeneity appropriate for crystallization.

PCT Kit comparison table.

PCT (with slides)

PCT (reagents only)

PCT (with plates and slides)

PCT (with plates and slides)

1. Crystallization and x-ray diffraction analysis of human CLEC-2. Aleksandra A. Watson and Christopher A. O’Callaghan. Acta Cryst. (2005). F61, 1094-1096.

Hampton Research, first in crystallization since 1991, developing and delivering crystallization and optimization screens, reagents, plates, and other tools for the crystallization of biological macromolecules, including proteins (antibody), peptides (insulin), and nucleic acids (DNA).

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