Run SEC on complex prior to crystallization to remove single entities, provided size difference makes sense. Not too many column options though.
Vaheh From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of careinaedgo...@yahoo.com Sent: Friday, November 24, 2023 4:00 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] What could these crystals be? I wanted to thank everyone for their suggestions and ideas regarding the eyeball shaped crystals that I got at the beginning of the month. I can confirm that these crystals do indeed contain protein and DNA which is good news. I have tried a number of different buffers and salts since then. I have also tried seeding but nothing I have tried has changed the morphology of the crystals. They remain thin, flat and eyeball/pumpkin seed shaped. It is not difficult to make the crystals, they form quite easily under quite a few conditions. The difficulty is in the diffraction. By changing the buffer conditions we can now see some very weak diffraction (previously there was no diffraction at all). Are there any suggestions as to how to improve diffraction of these crystals? I did try different cryoprotectants, parabar, glycerol, PEG but no difference. I think perhaps the problem is heterogeneity considering my sample contains both protein and DNA. Any suggestions or thoughts are welcome. Thank you Careina On Wednesday, November 8, 2023 at 06:18:46 PM GMT+2, careinaedgo...@yahoo.com<mailto:careinaedgo...@yahoo.com> <000002531c126adf-dmarc-requ...@jiscmail.ac.uk<mailto:000002531c126adf-dmarc-requ...@jiscmail.ac.uk>> wrote: The reservior solution is 0.2 M NaCl2, 0.1 M HEPES pH 7.5, and 25% 3350 PEG Protein buffer is 300 mM NaCl, 20 mM HEPES pH 7.5, 3mM TCEP The drop contains 1.5ul protein-DNA complex and 1.5 ul reservior solution On Wednesday, November 8, 2023 at 05:12:25 PM GMT+2, stephen.c...@rc-harwell.ac.uk<mailto:stephen.c...@rc-harwell.ac.uk> <stephen.c...@rc-harwell.ac.uk<mailto:stephen.c...@rc-harwell.ac.uk>> wrote: Hi Careina, Without knowing what's in your protein buffer or crystallisation condition it's hard to comment. Best wishes, Steve Dr Stephen Carr Research Complex at Harwell (RCaH) Rutherford Appleton Laboratory Harwell Oxford Didcot Oxon OX11 0FA United Kingdom Email stephen.c...@rc-harwell.ac.uk<mailto:stephen.c...@rc-harwell.ac.uk> tel 01235 567717 ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of careinaedgo...@yahoo.com<mailto:careinaedgo...@yahoo.com> <000002531c126adf-dmarc-requ...@jiscmail.ac.uk<mailto:000002531c126adf-dmarc-requ...@jiscmail.ac.uk>> Sent: 08 November 2023 15:00 To: ccp4bb <ccp4bb@jiscmail.ac.uk<mailto:ccp4bb@jiscmail.ac.uk>> Subject: [ccp4bb] What could these crystals be? Hi all We have been trying with no success to crystalize a protein. Recently we got these strange shape "crystals". They are hard and flat but they do not diffract at all. Any ideas as to what could cause this? Careina ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1<https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> This email and any attachments may contain confidential, copyrightand or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorized recipient of the addressee, please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to this email. Any views or opinions presented are solely those of the author and do not necessarily represent those of the Research Complex at Harwell. There is no guarantee that this email or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. 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