The primary screening variables are PEG, ion type, ionic strength, and pH. More than 60% of the published crystallizations utilized PEG as a primary crystallization reagent and in approximately 50% of those reports, the PEG was combined with an ion as a secondary crystallization reagent. PEG/Ion reagents are formulated without a buffer and are not pH titrated.

PEG/Ion 2 is an extension to the fundamental crystallization strategy in PEG/Ion. PEG/Ion 2 reagents cover the monodisperse, high purity Polyethylene glycol 3,350 and an array of neutralized and pH adjusted organic acids, multivalent ions, a novel Citrate BIS-TRIS propane buffer system and pH (4 – 8.8). The formulation of PEG/Ion 2 was developed at Hampton Research. Each of the 48 reagents in PEG/Ion 2 contains PEG 3,350 as the polymer (precipitant). The concentration of PEG is varied from 12% w/v to 20% w/v depending upon the type and concentration of buffer/salt paired with the polymer. Thirteen of the forty-eight PEG/Ion 2 reagents contain a separate buffer component. The remaining PEG/Ion 2 reagents are buffered by the titrated organic acid salt. Six of these thirteen conditions feature a novel Citric acid BIS-TRIS propane (CBTP) buffer. The CBTP buffer uses Citric acid and BIS-TRIS propane as the acid base pair to create a two component buffer system effective across pH 2.5 to 9.5. The ratio of Citric acid to BIS-TRIS propane determines the solution pH. Thirty-five of the forty-eight PEG/Ion 2 reagents contain a neutralized or pH adjusted organic acid in the presence of the polymer. Neutralized organic acids are highly effective crystallization salts.¹ Four PEG/Ion 2 reagents feature polyvalent cations. Two of these reagents contain cation mixes, saving sample by screening six different cations with only two reagents. Tryptone, a casein digest combinatorial library of peptides, is included in PEG/Ion 2. PEG/Ion 2 reagents 1-30, 42, 45-47 are formulated without a buffer and are not pH titrated.

PEG/Ion contains 48 unique reagents, 10 ml each.

PEG/Ion 2 contains 48 unique reagents, 10 ml each.

PEG/Ion HT contains 1 ml of each reagent from PEG/Ion and PEG/Ion 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.

PEG/Ion Screen

PEG/Ion 2 Screen

PEG/Ion HT

1. Expression, purification, crystallization and preliminary X-ray diffraction analysis of Bifidobacterium adolescentis xylose isomerase. C. V. dos Reis, A. Bernardes and I. Polikarpov. Acta Cryst. (2013). F69, 588-591.

2. A comparison of salts for the crystallization of macromolecules. Alexander McPherson. Protein Science (2001), 10:418-422.

3. Searching for silver bullets: An alternative strategy for crystallizing macromolecules. Alexander McPherson and Bob Cudney. Journal of Structural Biology Volume 156, Issue 3 , December 2006, Pages 387-406.

4. Mechanisms for Glycolipid Antigen-Driven Cytokine Polarization by Valpha14i NKT Cells. Sulliivan et al, The Journal of Immunology, 2010, 184, 141 -153. (PEG/Ion 2)

5. Optimization of salt concentration in PEG-based crystallization solutions. Yamanaka M, Inaka K, Furubayashi N, Matsushima M, Takahashi S, Tanaka H, Sano S, Sato M, Kobayashi T, Tanaka T. J Synchrotron Radiat. 2011 Jan;18(1):84-7. doi: 10.1107/S0909049510035995. Epub 2010 Nov 5.

6. Crystallization and preliminary X-ray crystallographic analysis of importin-[alpha] from Neurospora crassa. N. E. Bernardes, A. A. S. Takeda, F. Z. Freitas, M. C. Bertolini and M. R. M. Fontes. Acta Cryst. (2014). F70, 501-504 [ doi:10.1107/S2053230X14005068 ]

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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Most G protein-coupled receptors (GPCRs) have been crystallized in Polyethylene glycol (PEG) 400 based reagents¹. PEG/Ion 400 is designed as a 96 reagent crystallization screen that is compatible with the Lipidic Cubic Phase (LCP).

PEG/Ion 400 is supplied in a sterile, polypropylene 96 Deep Well block, each reservoir containing 1 ml of sterile filtered reagent. The block is compatible with robotic and multi-channel pipet liquid handling systems and is heat sealed using a special polypropylene backed film. Each PEG/Ion 400 is supplied with an adhesive sealing film which can be used to seal the block after removing the heat seal.

The screen combines a single concentration (30% v/v) of high purity Polyethylene glycol 400 and 48 different high purity salts, comprising both anions (malate, malonate, nitrate, phosphate, succinate, sulfate, tartrate, and thiocyanate) and cations (ammonium, cadmium, calcium, ethylammonium, lithium, magnesium, nickel, potassium, sodium, tetraethylammonium, and zinc) in two concentrations (0.1 and 0.4 M) which due to their unique pH characteristics also affords a reasonable pH screen (approximate pH range of 4 to 9.5). The primary screen variables are PEG, ion type, ionic strength, salt concentration, and pH.

PEG/Ion 400

1. Successful Strategies to Determine High-Resolution Structures of GPCRs. Xiang J, Chun E, Liu C, Jing L, Al-Sahouri Z, Zhu L, Liu W. Trends Pharmacol Sci. 2016 Dec;37(12):1055-1069.

2. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Kang Y, Zhou XE, Gao X, et al. Nature. 2015;523(7562):561-567. doi:10.1038/nature14656.

3. Development of an Automated High Throughput LCP-FRAP Assay to Guide Membrane Protein Crystallization in Lipid Mesophases. Fei Xu, Wei Liu, Michael A. Hanson, Raymond C. Stevens, and Vadim Cherezov. Cryst. Growth Des., 2011, 11 (4), pp 1193-1201.

4. A comprehensive review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes. Martin Caffrey. Acta Crystallogr F Struct Biol Commun. 2015 Jan 1; 71(Pt 1): 3-18.

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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It is designed to evaluate polymer based crystallization reagents and pH in low (PEGRx 1) to medium ionic strength (PEGRx 2). It is also designed for use as a secondary or optimization screen to follow the Hampton Research Index screen when polymer based reagents produce hits and interesting solubility leads.

PEGRx 1 is a crystallization reagent kit designed to evaluate an array of polymers of varying molecular weight in a low ionic strength environment versus a wide range of pH. Polymer reagents include Polyethylene glycols, Polyethylene glycol monomethyl ethers, and Jeffamines^®. The molecular weight range between 200 and 20,000 is evaluated in a low ionic strength formulation. Ten different buffers are used to span the range of pH between 3.5 and 9. The primary screen variables are polymer type, polymer molecular weight, pH and low ionic strength.

PEGRx 2 is a crystallization reagent kit designed to evaluate an array of polymers of varying molecular weight in a medium ionic strength environment in the presence of additives, salts, volatile organics and polyols versus a wide range of pH. Polymer reagents include Polyethylene glycols and Polyethylene glycol monomethyl ethers. The polymer molecular weight range between 200 and 20,000 is evaluated in a medium ionic strength formulation. Ten different buffers are used to span the range of pH between 3.5 and 9. The primary screen variables are polymer type, polymer molecular weight, pH and secondary reagents which include additives, salts, volatile organics and polyols.

PEGRx 1 contains 48 unique reagents, 10 ml each.

PEGRx 2 contains 48 unique reagents, 10 ml each.

PEGRx HT contains the 96 PEGRx 1 and PEGRx 2 reagent 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.

PEGRx 1

PEGRx 2

PEGRx HT

1. Crystallization and preliminary X-ray crystallographic studies of DesR, a thermosensing response regulator in a two-component signalling system from Streptococcus pneumoniae. Ae Kyung Park, Seung Min Bong, Jin Ho Moon and Young Min Chi. Acta Cryst. (2009). F65.

2. Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors.  Zhang L, Lin D, Sun X, Curth U, Drosten C, Sauerhering L, Becker S, Rox K, Hilgenfeld R. Science. 2020 Apr 24;368(6489):409-412. doi: 10.1126/science.abb3405. Epub 2020 Mar 20.

3. Structural insight into the electron transfer pathway of a self-sufficient P450 monooxygenase.  Zhang, L., Xie, Z., Liu, Z. et al.  Nat Commun 11, 2676 (2020). https://doi.org/10.1038/s41467-020-16500-5.

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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Proteases can be used to generate small, active fragments or domains of the target protein for crystallization.⁹ The fragment or domain can be used directly for crystallization experiments. Or the proteolytic sample analyzed by gel electrophoresis and/or mass spectrometry for mass and sequence for subsequent cloning, expression, purification and crystallization. Using proteolysis to enhance sample crystallization, the current overall success rate for yielding a deposited crystal structure is currently better than 12%.³

Proti-Ace (HR2-429) contains three aliquots of 6 unique proteases (a-Chymotrypsin, Trypsin, Elastase, Papain, Subtilisin and Endoproteinase Glu-C) and six aliquots of Proti-Ace Dilution Buffer. Each protease is supplied in a stable, lyophilized format in an optimized digest buffer. Simply add water when ready to use.

Proti-Ace 2 (HR2-432) contains three aliquots of 6 unique proteases (Proteinase K, Clostripain (Endoproteinase-Arg-C), Pepsin, Thermolysin, Bromelain and Actinase E) and six aliquots of Proti-Ace Dilution Buffer. Each protease is supplied in a stable, lyophilized format in an optimized digest buffer. Simply add water when ready to use.

The unique freeze dried formulation of the Proti-Ace kits offers a much improved protease stability compared to liquid protease formulations.

Proti-Ace™

Proti-Ace™ 2

1. Allan D′Arcy, personal communication, 1989-2009.

2. In situ proteolysis for protein crystallization and structure determination. Dong, A et al. Nature Methods – 4, 1019 – 1021 (2007).

3. In Situ Proteolysis to Generate Crystals for Structure Determination: An Update. Amy Wernimont, Aled Edwards. PLoS ONE 4(4): e5094. doi:10.1371/ journal.pone.0005094.

4. The use of in situ proteolysis in the crystallization of murine CstF-77. Tong et al. Acta Cryst. (2007). F63, 135-138.

5. A brief history of protein crystal growth. McPherson, A. Journal of Crystal Growth, vol. 110, issue 1-2, pp. 1-10, 1991.

6. Preparation and analysis of protein crystals. McPherson, A. John Wiley & Sons, Inc. 1982. ISBN 089464355X.

7. A crystallizable form of the Streptococcus gordonii surface antigen SspB C-domain obtained by limited proteolysis. Forsgren et al. Acta Cryst. (2009). F65, 712-714.

8. Preliminary X-ray analysis of a human VH fragment at 1.8 angstrom resolution. Gaur, Kupper, Fischer & Hoffman. Acta Cryst. (2004). D60, 965-967.

9. Replication Protein A Characterization and Crystallization of the DNA Binding Domain. Pfuetzner et al. The Journal of Biological Chemistry, Vol. 272, No. 1, Issue of January 3, pp. 430-434, 1997.

10. Combining in situ proteolysis and mass spectrometry to crystallize Escherichia coli PgaB. Little et al. Acta Cryst. (2012) F68, 842-845.

11. Proteolysis of Native Proteins – Trapping of a Reaction Intermediate. Chenyi Wu, Duncan H. L. Robertson, Simon J. Hubbard, Simon J. Gaskell and Robert J. Beynon. doi: 10.1074/jbc.274.2.1108 January 8, 1999 The Journal of Biological Chemistry, 274, 1108-1115.

12. Crystallization of mouse RIG-I ATPase domain: in situ proteolysis. Civril F, Hopfner KP. Methods Mol Biol. 2014;1169:27-35. doi: 10.1007/978-1-4939-0882-0_3.

13. Crystallization and preliminary X-ray crystallographic analysis of YfcM: an important factor for EF-P hydroxylation. K. Kobayashi, T. Suzuki, N. Dohmae, R. Ishitani and O. Nureki. Acta Cryst. (2014). F70, 1236-1239 [ doi:10.1107/S2053230X14015726 ]. Synopsis: in situ proteolysis crystallization method produces crystals diffracting to 1.45 Å

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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Salt is the only primary crystallization reagent (precipitant) utilized. Based on a design of 96 conditions, the screen evaluates a broad portfolio of crystallization salts of varying concentration and pH. The selection, concentration, and pH of the salts were determined by data mining the BMCD¹ and other crystallization databases, crystallization reports in the literature, and internal crystallization trials performed at Hampton Research. Based on this analysis, up to 35% of protein crystallizations involve salt as the primary crystallization reagent. SaltRx can be used as a primary crystallization screen when salt, ionic strength and pH are desired or suspected as appropriate crystallization variables. It is also useful as a secondary screen when salt only reagents/conditions from screens such as Index™, Crystal Screen™, and Grid Screen™ produce crystals and when further screening for additional salt conditions or optimization is desired. Since this screen does not contain volatile organics, it is compatible with microbatch, vapor diffusion, liquid and gel diffusion crystallization methods. SaltRx may also be used for dialysis crystallization in conjunction with Dialysis Buttons™.

The original SaltRx kit (HR2-108) contained 96 unique reagents, 10 ml each. This kit has been discontinued and converted into two 48 reagent based kits, SaltRx 1 and SaltRx 2, available separately, which when used together are identical to the original SaltRx.

SaltRx 1 contains 48 unique reagents, 10 ml each. SaltRx 1 contains reagents 1-48 of the original SaltRx 96 reagent kit.

Salt Rx 2 contains 48 unique reagents, 10 ml each. SaltRx 2 contains reagent 49-96 of the original SaltRx 96 reagent kit.

SaltRx HT contains the 96 SaltRx 1 and SaltRx 2 reagents 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.

Measured pH range of kit is 4.1 to 9.0 at 25°C
Average measured pH of kit is 6.9 at 25°C
Median measured pH of kit is 7.3 at 25°C
Mode measured pH of kit is 7.4 at 25°C

SaltRx 1

SaltRx 2

SaltRx HT

1. Cloning, expression, purification and preliminary X-ray diffraction studies of a mycobacterial protein implicated in bacterial survival in macrophages. A. E. Shahine, P. Y. Chan, D. Littler, J. Vivian, R. Brammananth, P. K. Crellin, R. L. Coppel, J. Rossjohn and T. Beddoe. Acta Cryst. (2013). F69, 566-569.

2. Gilliland, G.L., Tung, M., Blakeslee, D.M. and Ladner, J. 1994. The Biological Macromolecule Crystallization Database, Version 3.0: New Features, Data, and the NASA Archive for Protein Crystal Growth Data. Acta Crystallogr. D50 408-413.

3. Crystallization and preliminary crystallographic analysis of molybdenum-cofactor biosynthesis protein C from Thermus thermophilus. S. P. Kanaujia, C. V. Ranjani, J. Jeyakanthan, S. Baba, L. Chen, Z.-J. Liu, B.-C. Wang, M. Nishida, A. Ebihara, A. Shinkai, S. Kuramitsu, Y. Shiro, K. Sekar and S. Yokoyama. Acta Cryst. (2007). F63, 27-29.

4. Crystal-contact engineering to obtain a crystal form of the Kelch domain of human Keap1 suitable for ligand-soaking experiments. Stefan Horer, Dirk Reinert, Katja Ostmann, Yvette Hoevels and Herbert Nar. Acta Cryst. (2013). F69, 592-596.

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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