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Maria, >From my experience with co-crystallization experiments, if crystals appear >immediately after setting up the drop in presence of small molecules, they most likely are crystals of small molecules and not of protein. Since you have 8% 8K PEG, initially the solution is heterogeneous with pockets of high viscosity that will precipitate the small molecules. Over the period of time, solution becomes homogenous that may dissolve the crystals. To rule out this, set up two crystallization experiments under similar conditions. 1) only the protein 2) only the adenosine Regards Anthony ----------------------------------------------------- Dr. Anthony Addlagatta Center for Chemical Biology Indian Institute of Chemical Technology [IICT] Tarnaka, Hyderabad AP-500 607, INDIA Tel:91-40-27191812 Web: https://sites.google.com/site/chembioliict/home/dr-anthony-addlagatta-1 ---------- Original Message ----------- From: dusky dew <duskyde...@gmail.com> To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 7 May 2014 00:52:22 -0700 Subject: Re: [ccp4bb] Crystals Disappearing Overnight > *********************** > This message has been scanned by the InterScan for CSC SSM at IICT and found > to be free of known security risks. > *********************** > > 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> 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 > > > > Web www.hamptonresearch.com > > > > > > > > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > dusky dew > > Sent: Saturday, May 03, 2014 1:29 AM > > To: 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> 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 > >> > >> Web www.hamptonresearch.com > >> > >> > >> > >> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > dusky dew > >> Sent: Friday, May 02, 2014 4:39 AM > >> To: 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 > >> > >> > >> > >> ------- End of Original Message ------- ------------------------------------------------------------------------------------------------- Note: The information contained in the e-Mail message and/or attachments to it may contain confidential or privileged information. If you are not the intended recipient, any dissemination, use, review, distribute, prinitng or copying of the information contianed in this e-Mail message and/or attachments to it are strictly prohibited. If you have received this communication in error. Please notify us by reply e-Mail or telephone and immediately and permanently delete the message and any attachment. Thank you.
