Hi all Just wanted to thank you all for all the suggestions and tell you of all the things i tried what finally worked, if anyones interested.
Among the several things mentioned here, the crystals diffracted the best (~ 2.9 Å at the synchrotron) when grown in a solution containing either 5% ethylene glycol and cryo protected in 20% ethylene glycol or 15% glycerol and protected in 25% glycerol. Thanks for all the help Mahesh On Thu, Jan 23, 2014 at 7:04 AM, Elizabeth Morris < [email protected]> wrote: > Hi Mahesh, > > I have experienced similar situations with AmSO4 as a precipitant. One > thing I found worked is to optimise crystallization in a lower > concentration of AmSO4 so that you reduce the amount of salt crystals you > get when you try and loop your crystals. I got hits in 2 M AmSO4, but I > have managed to reduce this to ~1 M and still get crystals by: > > - increasing protein concentration > - varying pH/buffer > - using microseeding to obtain crystals > > I have less experience with different types of cryoprotectants, but in my > opinion, if you problem is that you see salt crystals when you open your > droplet to the air, try reducing the salt concentration in your drop. > > Alternatively, perhaps your microscope is heating up your sample and > increasing the rate of evaporation from your drop. If that is the case, try > to work at lower intensities of the lamp, or turn it off more frequently. > > Finally, I have heard of people putting a damp tissue or a humidifier near > to where you are looping your crystals to reduce evaporation of water from > your drop. > > Good luck! > > Liz > > > > > > > Quoting Mahesh Lingaraju <[email protected]> on Wed, 22 Jan 2014 12:28:15 > -0500: > > Hello folks, >> >> I have crystals for a protein which form in 0.1 M MES pH 6-6.75, 10% >> dioxane and 1.6-2 M Ammonium sulphate. Based on little experience I have >> finding the right cryoprotection for salt based conditions; I have tried >> glycerol (15-25%), Ethylene glycol (20-25%), DMSO (15-20%) and increasing >> ammonium sulphate concentration in presence of 5-10% glycerol. The >> crystals >> disintegrate in any kind of PEG based cryo. I made all these solutions in >> the mother liquor and tested if they freeze clearly before using them. >> However when I loop the crystals and try to soak them in these cryo- >> mother >> liquor, a lot of salt crystals suddenly form around the protein crystal >> and >> I see diffraction only from these salt crystals. The best I have been able >> to get so far is ~ 9Å diffraction (Home-source) with DMSO as the >> cryoprotectant. >> >> I have also tried using 1 M sodium malonate as the cryoprotectant but my >> crystals are not too stable in this mother liquor probably because I had >> to >> lower the ammonium sulphate by ~ 7-10% to make the drop not form salt >> crystals instantly when exposed to air. >> >> Other than trying to make the crystals more cryo-ready by finding other >> hits or may be growing the same crystals with some cryoprotectant, I was >> wondering if any of you have ideas based on your experience or suggestions >> in case I am doing everything wrong in the first place. >> >> Thanks for all the help, >> >> Mahesh >> >> > > > -- > The University of Edinburgh is a charitable body, registered in > Scotland, with registration number SC005336. > > >
