Hi.
I'm not very sure what you mean by stabilising solution. I assume cryo protectant. I have successfully soaked crystals with different sugars and I find the best cryo is the actual sugar you use. I've used glucose and maltose at around 25% and they work brilliant as cryos. Things like galactose need to be warm or they do not stay soluble and this can be a problem. My suggestion would be to use your HEPES condition with added sugar at lower concentration 10, 20, 50 mM, . and after a soaking period, stick them into a high sugar cryo buffer for a few seconds and the freeze them. I also think that you probably don't need such a long soaking time. I find that I get perfectly occupied sites after a couple of hours. If anything, if you leave them too long you may get the substrate coming out of the active site. Also, certain cryos like glycerol can compete with you substrate and kick it out. Your soak could not only destroy the crystal but it can also change your space group and what happens is that you need to leave enough time to let your crystal repack or you may get problems trying to index them. Hope that helps Juan Hello everyone, I am asking for some help with my soaking experiment, my protein should bind with oligosaccharide such as maltotriose,tetraose,... the crystalization condition is 0.1 M HEPES, ph 7.2. I am having trouble finding stabilization buffer: it does not like PEG, ( it dissolved in PEG solution faster than in pure water) , I also tried glycerol, which is not better. I finally use 10% MPD, but when I add ligand ( oligosaccharide), from several mM to 50 mM , the crystals can only last from 12 hrs to 24 hrs before crack. this make the soaking really hard. by the way,it seems that the crack is associated with adding ligand, ( without ligand,it could last several days), is that because the binding destroyed the crystal? anyone have suggestion for this situation ? Thanks a lot
