Re: [ccp4bb] crystallization with hydrophobic ligands
Using mixed solvents is another approach: Ciccone L., Tepshi L., Nencetti, S. Stura E.A. (2014) Transthyretin complexes with curcumin and bromo-estradiol: Evaluation of solubilizing multicomponent mixtures New Biotech. 32:54–64 http://dx.doi.org/10.1016/j.nbt.2014.09.002 Volatile solvents are a problem when you try to harvest the crystals. Enrico. On Wed, 15 Oct 2014 20:35:33 +0200, Jurgen Bosch jbos...@jhu.edu wrote: An alternative is to dissolve your compound in MeOH and dispense it either manually or via robot, let the plate sit sometime in the hood for faster evaporation and then add your protein + reservoir. Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742tel:%2B1-410-614-4742 Lab: +1-410-614-4894tel:%2B1-410-614-4894 Fax: +1-410-955-2926tel:%2B1-410-955-2926 http://lupo.jhsph.edu On Oct 15, 2014, at 5:18 PM, Keller, Jacob kell...@janelia.hhmi.orgmailto:kell...@janelia.hhmi.org wrote: Since you mentioned EtOH, why not do this: -Make a tray with the appropriate mother liquors -Make a drop for each well containing mother liquor and high-concentration ligand in EtOH (you could vary the ratios here as needed.) -Equilibrate by vapor diffusion until EtOH all goes into the well soln (couple of hours at the most?) -Add protein to these drops You could skip right to the protein step if your protein doesn't mind EtOH at fairly high concentrations, and anyway it will be gone fairly quickly, esp at RT Jacob Keller From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] on behalf of Monica Mittal [monica.mitta...@gmail.commailto:monica.mitta...@gmail.com] Sent: Wednesday, October 15, 2014 2:13 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystallization with hydrophobic ligands Dear All Can anyone give suggestions for handling the solubility problem of highly hydrophobic compounds, during co-crystallization or inhibition assays? The ligands I am using are almost insoluble in aquous medium. In DMSO or 95% Ethanol, the solubility is higher. Besides crystallization, this solubility is also a hindrance for in-vitro or in-vivo assays requiring higher conc. of ligand. Thanks in advance ! Monica -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
[ccp4bb] crystallization with hydrophobic ligands
Dear All Can anyone give suggestions for handling the solubility problem of highly hydrophobic compounds, during co-crystallization or inhibition assays? The ligands I am using are almost insoluble in aquous medium. In DMSO or 95% Ethanol, the solubility is higher. Besides crystallization, this solubility is also a hindrance for in-vitro or in-vivo assays requiring higher conc. of ligand. Thanks in advance ! Monica
Re: [ccp4bb] crystallization with hydrophobic ligands
Yes, this is tough. We mostly have used DMSO or DMF. You can try detergents, but they tend not to be that effective in solubilisation and they might bind to your protein rather than the compound you 'd like to bind. If you'd like to be adventurous you could try using cyclodextrins as a solubilisation vehicle. These cyclic sugars are often used industrially to get hydrophobic compounds soluble in water. If your protein has high enough affinity for your ligand this approach may well work. Some pioneering may be required though Good luck, Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Monica Mittal [monica.mitta...@gmail.com] Sent: Wednesday, October 15, 2014 2:13 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystallization with hydrophobic ligands Dear All Can anyone give suggestions for handling the solubility problem of highly hydrophobic compounds, during co-crystallization or inhibition assays? The ligands I am using are almost insoluble in aquous medium. In DMSO or 95% Ethanol, the solubility is higher. Besides crystallization, this solubility is also a hindrance for in-vitro or in-vivo assays requiring higher conc. of ligand. Thanks in advance ! Monica
Re: [ccp4bb] crystallization with hydrophobic ligands
Monica, I struggled with this a lot too. I had some success with low molecular weight PEG (100-400) as a solvent, its well worth a try. -bob On Wed, Oct 15, 2014 at 9:13 AM, Monica Mittal monica.mitta...@gmail.com wrote: Dear All Can anyone give suggestions for handling the solubility problem of highly hydrophobic compounds, during co-crystallization or inhibition assays? The ligands I am using are almost insoluble in aquous medium. In DMSO or 95% Ethanol, the solubility is higher. Besides crystallization, this solubility is also a hindrance for in-vitro or in-vivo assays requiring higher conc. of ligand. Thanks in advance ! Monica
Re: [ccp4bb] crystallization with hydrophobic ligands
Since you mentioned EtOH, why not do this: -Make a tray with the appropriate mother liquors -Make a drop for each well containing mother liquor and high-concentration ligand in EtOH (you could vary the ratios here as needed.) -Equilibrate by vapor diffusion until EtOH all goes into the well soln (couple of hours at the most?) -Add protein to these drops You could skip right to the protein step if your protein doesn't mind EtOH at fairly high concentrations, and anyway it will be gone fairly quickly, esp at RT Jacob Keller From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Monica Mittal [monica.mitta...@gmail.com] Sent: Wednesday, October 15, 2014 2:13 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystallization with hydrophobic ligands Dear All Can anyone give suggestions for handling the solubility problem of highly hydrophobic compounds, during co-crystallization or inhibition assays? The ligands I am using are almost insoluble in aquous medium. In DMSO or 95% Ethanol, the solubility is higher. Besides crystallization, this solubility is also a hindrance for in-vitro or in-vivo assays requiring higher conc. of ligand. Thanks in advance ! Monica
Re: [ccp4bb] crystallization with hydrophobic ligands
An alternative is to dissolve your compound in MeOH and dispense it either manually or via robot, let the plate sit sometime in the hood for faster evaporation and then add your protein + reservoir. Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742tel:%2B1-410-614-4742 Lab: +1-410-614-4894tel:%2B1-410-614-4894 Fax: +1-410-955-2926tel:%2B1-410-955-2926 http://lupo.jhsph.edu On Oct 15, 2014, at 5:18 PM, Keller, Jacob kell...@janelia.hhmi.orgmailto:kell...@janelia.hhmi.org wrote: Since you mentioned EtOH, why not do this: -Make a tray with the appropriate mother liquors -Make a drop for each well containing mother liquor and high-concentration ligand in EtOH (you could vary the ratios here as needed.) -Equilibrate by vapor diffusion until EtOH all goes into the well soln (couple of hours at the most?) -Add protein to these drops You could skip right to the protein step if your protein doesn't mind EtOH at fairly high concentrations, and anyway it will be gone fairly quickly, esp at RT Jacob Keller From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] on behalf of Monica Mittal [monica.mitta...@gmail.commailto:monica.mitta...@gmail.com] Sent: Wednesday, October 15, 2014 2:13 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystallization with hydrophobic ligands Dear All Can anyone give suggestions for handling the solubility problem of highly hydrophobic compounds, during co-crystallization or inhibition assays? The ligands I am using are almost insoluble in aquous medium. In DMSO or 95% Ethanol, the solubility is higher. Besides crystallization, this solubility is also a hindrance for in-vitro or in-vivo assays requiring higher conc. of ligand. Thanks in advance ! Monica
