Dear Maria have you collected the crystals before they dissolve and washed them, then either dissolved them in SDS PAGE buffer and ran them on an SDS PAGE gel (for say a western blot) or capillary mounted them, then shot them on the x-ray set to determine if they are protein or small molecule, or tried to freeze them, or washed them and sent them to mass spec to determine if they are protein?
If the crystals are protein try different conditions, such as a gradient of PEG vs NaCl, seeding using the crystals crushed before they dissolve, protein concentration, ligand concentration etc and maybe a different temperature. Good luck G On May 7, 2014, at 1:52 AM, dusky dew wrote: I tried microbatch and the crystals are not stable. They dissolve overnight. I also have reproducibility issue. Can this be due to poor stability of adenosine? Best Maria On Monday, May 5, 2014, Bob Cudney <b...@hrmail.com<mailto:b...@hrmail.com>> wrote: > Try the microbatch first to see if the problem is related to ionic strength. > > > > When possible and practical it is good to change only one variable at a time > to identify cause and effect. > > > > Kind Regards, Bob Cudney > > > > Hampton Research > > 34 Journey > > Aliso Viejo, CA 92656-3317 USA > > > > Telephone 1 949 425 1321 Extension 200 > > Fax 1 949 425 1611 > > E-mail b...@hrmail.com<mailto:b...@hrmail.com> > > Web www.hamptonresearch.com<http://www.hamptonresearch.com/> > > > > From: CCP4 bulletin board > [mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of > dusky dew > Sent: Saturday, May 03, 2014 1:29 AM > To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> > Subject: Re: [ccp4bb] Crystals Disappearing Overnight > > > > Thank you all for getting back! > > I will set up the drops using microbatch method. > Regarding the temp, I set them up in lab and put them in incubator. The lab > temp may be slightly higher. So are they not stable at lower temp? Or its the > shock? > So how can I take care of the temp issue? > > Thanks again! > Maria > > On Saturday, May 3, 2014, Bob Cudney > <b...@hrmail.com<mailto:b...@hrmail.com>> wrote: >> Hello Maria, >> >> >> >> Check to see if there might have been a temperature change between the time >> the crystals were present and when the crystals disappeared. If your sample >> has temperature dependent solubility, in this relatively low ionic strength >> condition, a temperature change could mean the difference between the >> presence and absence of crystals. That being said, if the experiment is >> returned to the temperature that produced the crystals, the crystals >> should/might reappear. >> >> >> >> If your drop is made by mixing 1 part of protein with 1 part of reagent the >> initial drop concentration would be 10 mM Tris, 150 mM NaCl, 2.5% w/v PEG >> 8,000, 50 mM Sodium cacodylate. Is this a vapor diffusion experiment? If >> yes, then the reservoir would be 5% w/v PEG 8,000, 100 mM Sodium cacodylate. >> The ionic strength of your drop would initially be higher than the ionic >> strength in your reservoir. This means water vapor leaves the reservoir and >> vapor diffuses into the drop, lowering the protein and reagent concentration >> in your drop. This decrease in relative supersaturation could dissolve a >> crystal. Your set up would be a reserve vapor diffusion. You say the >> crystals appeared right after setting the experiment so your crystallization >> is essentially a batch experiment. Therefore you might want to change your >> set up from a vapor diffusion to a microbatch experiment under oil. If you >> need more information about how to perform a microbatch experiment, let me >> know and I’ll explain. >> >> >> >> Hope this helps. >> >> >> >> Kind Regards, Bob Cudney >> >> >> >> Hampton Research >> >> 34 Journey >> >> Aliso Viejo, CA 92656-3317 USA >> >> >> >> Telephone 1 949 425 1321 Extension 200 >> >> Fax 1 949 425 1611 >> >> E-mail b...@hrmail.com<mailto:b...@hrmail.com> >> >> Web www.hamptonresearch.com<http://www.hamptonresearch.com/> >> >> >> >> From: CCP4 bulletin board >> [mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of >> dusky dew >> Sent: Friday, May 02, 2014 4:39 AM >> To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> >> Subject: [ccp4bb] Crystals Disappearing Overnight >> >> >> >> Dear All, >> >> >> >> I am trying to crystallize a protein with Adenosine. My protein is in 20 mM >> Tris, 300 mM NaCl and the crystals appear in a condition with 5 percent >> PEG8K, 0.1 M Sodium Cacodylate. The protein is incubated with adenosine for >> 1/2 hr before setting the drop. The crystals appear right after the drop is >> set but unfortunately they dissolve overnight. The plate is kept at 16 >> degree. >> >> >> >> Could anyone elaborate on this. Is it possibly occurring because Adenosine >> has stability issues. >> >> >> >> Thanks for your suggestions. >> >> ~ Maria >> >> >> >>
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