Jonathan, While your claim of oxidative degradation of PEG1000 may be true -- I gather you mean that the conversion of the ends of the PEG polymers to aldehydes or peroxides, then to carboxylates -- you should check out Fran Jurnak’s old paper (Journal of Crystal Growth 76, 577, 1986). The synthesis of PEG often contains some of phosphoric acid due to the way they terminated the chain elongation. It is variable from batch to batch and from supplier to supplier; Merck (Germany) is a fairly good source, but Baker/Union Carbide isn't. Unless Jena took the time and effort to purify the PEG (see Bill Ray's article in the same issue, p. 562), you have another factor that can drop the pH. You might ask where Jena buys their PEG stock.
Also, the way many companies make the screens are not always clear. Some just mix stocks, meaning the pH of the Tris stock (perhaps at 1 M) was pH 8, but when diluted down with the other components to make the 150 mM concentration for the screen mixture, the pH can be significantly different. The dilution of Tris would drop the pH. Cheers, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: rmgarav...@gmail.com **************************************************************** > On Jul 31, 2017, at 8:34 AM, Jonathan Bailey <baile...@tcd.ie> wrote: > > Dear CCP4bb community > > > > I apologies for the slightly off topic post. > > > > We have recently had success crystallizing a membrane protein (diffraction > > 3 Å at a synchrotron source) using the in meso method, the hit condition was > from the Jena Bioscience screen Pi-minimal condition number #57. > > > > Hit condition – 47.1 % w/v PEG1000, 150 mM Tris pH 8.0, 80 mM Potassium > bromide > > > > > The screen is old and expired 12/20/2013 (lot # JBS00013133), we have tried > to reproduce the crystals using homemade optimization screens around the hit > condition but have not had any success. We have tried reproducing the hit > using a new (not expired) Pi-minimal screen but had no success. We are only > able to reproduce the crystals using the expired screen and we do not have > much of it left. > > > We went back and tested the pH of the condition that had given crystals, the > expected pH was 7.9 but we found it to be 6 – 6.5 using a pH indicator strip. > We believe the drop in pH is caused by oxidative degradation of the PEG1000 > resulting in the formation of carboxylic acid species. > > > > We have contacted Jena Bioscience to try and get some of the old screen stock > but unfortunately they do not have any. > > > > My question is does anyone out there happen to have any expired screen stocks > of this Pi-minimal condition (#57), ideally from the same lot (lot # > JB200013133), that they would be willing to send us. > > > Does anyone have any advice as to how to reproduce the condition? We’ve > considered bubbling oxygen through and heating the sample to accelerate the > oxidation process. > > > King Regards > > > > Jonathan Bailey (PhD student) > > > > Professor Martin Caffrey Lab MS&FB group Trinity College Dublin >
