Hi Jonathan,

 Old buffer may have slow evaporation with some side reactions. The concentration of each component may increase a little bit.  In this case,  I would consider the condition as the origin for further optimization. Each component may need to be considered separately.

For 150mM Tris pH 8.000, 
The pH and concentration may  have a slight change (increase ?).  I will try the concentration of 150mM, 155 mM and 160mM. For pH,  pH 8.1 and pH 8.2...

For KBr, the concentration may be 80, 85, 90mM or higher.

For 47.1% w/v PEG1K (Pretty high concentration), 
As the result for ring-opening epoxide reaction, it is not very stable anyway. The reaction always continued. Basic condition made the case even worse. In this case, the Mn (MW) of PEG1K may not be that average anymore. It is very possible that the polymer chain is elongated. Of course, the concentration of PEG is increased too. In the meanwhile, the presence of KBr may cause further chemical modification on the PEG chains.  You may try PEG 95--1050 (Sigma P3515), PEG 1305-1595 (Sigma 202136), PEG3K or PEG 3350, etc.  


Best,

Kevin

P.S. If you take a look from the top of your old solution in the tube, what's the color? Slight Yellow? You can use a tube with dd water a reference. 


On Mon, Jul 31, 2017 at 5:34 AM, Jonathan Bailey <[log in to unmask]> 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 




--
Kevin Jin
 
Sharing knowledge each other is always very joyful......
 
Website: http://www.jinkai.org/