Dear William, When in doubt, I would usually harvest a few needle clusters (especially as it sounds like you got plenty now), wash them with crystallization reservoir and then run them on SDS-PAGE gel. If you see a band of the right molecular weight you have most probably crystallized your protein of interest.
HTH, Best, D ________________________________________ From: [email protected] [[email protected]] Sent: 11 April 2014 08:47 To: ccp4bb Subject: [ccp4bb] AW: [ccp4bb] suggestion on improving sea urchin like crystals Dear William, The lowest resolution spots you can see tell you something about your cell dimensions since the resolution cannot be lower than the longest distance in your unit cell. If you see spots at <30Å resolution, you are pretty sure you have protein crystals, if you only see spots at 10-15Å, you probably have crystals of something else. However, it looks like you have big problems with freezing, there are strong ice/salt rings and the freezing may have done a lot of damage. I would recommend to take a few shots at room temperature as well to see the native diffraction. If the shots were at room temperature, you have a problem with salt crystallizing out, probable the moment you open the well and some solvent evaporates. In this case, I would try to find some suitable cryobuffer, which you could add to the drop the moment you open the well. Finally, if you are sure your crystals are protein, you could grind some crystals and use them as seeds in a crystallization screen. Good luck! Herman Von: CCP4 bulletin board [mailto:[email protected]] Im Auftrag von william lee Gesendet: Freitag, 11. April 2014 04:03 An: [email protected] Betreff: Re: [ccp4bb] suggestion on improving sea urchin like crystals Here is some information regarding to my crystals. Crystallization condition: · 0.9M-1.7M Ammonium Tartrate Dibasic · 50mM-150mM Bis-Tris Propane (pH7.0 & 6.5) / 50mM-150mM Tris (pH7.5 & 8.5) Protein buffer contains 50mM ADA, pH6.5, 100mM NaCl, 10mM B-ME Regards William ________________________________ Date: Fri, 11 Apr 2014 01:50:18 +0100 From: [email protected]<mailto:[email protected]> Subject: [ccp4bb] suggestion on improving sea urchin like crystals To: [email protected]<mailto:[email protected]> Dear All, I am currently working on a ligand bound protein complex. From my initial crystallization screens I have identified a condition which gives me sea urchin like crystals. I managed to repeat these crystals in my optimization conditions, in fact I can see crystals in all the wells but there is no significant difference between them. All conditions give me similar size and amount of crystals. To confirm these are protein crystals or not, I tried expose the crystals to X-ray beam (in-house). Good news is that I have no obvious salt diffraction at high resolution, but the bad news is my low resolution diffractions are not really believable. In addition, these crystals seem to be quite easy to separate into needles but they are too small to tell if they do crack like most the protein crystals. I have attached a picture of my crystals and the diffraction pattern I got at low resolution. I am hoping if anyone can suggest me on how to improve or change the shape of these crystals if they are genuine protein crystals. Many thanks Kind regards William -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised 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 the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail 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. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
