Hi, Another way of estimating a starting protein concentration is to watch your concentration process – if your protein is in a spin concentrator (with an appropriate membrane cutoff size say ~ [MW protein]/3) and is losing volume really quickly then keep going. As soon as the concentration starts slowing down try that concentration. Actually, you should really follow the experience of the Oxford SGC where they always set up three drop ratios of protein to reservoir solution – 1:2, 1:1 and 2:1.
Cheers, Janet From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Edward Snell Sent: Thursday, 9 January 2020 3:52 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Protein concentration for the initial crystallisation trials Hi Armando, I echo Rodger’s advice at 10 mg/ml to start. Our high-throughput crystallization lab has a FAQ page that notes this https://hwi.buffalo.edu/high-throughput-crystallization-center/hts-faqs/ but we absolutely recommend looking at conditions where the precipitant concentration varies AND doing a screen with a different protein concentration if sample is available. Both have considerable impact on outcome and the screens we use are designed to provide information on this if they are interpreted correctly. There are a lot of internal references available at https://hwi.buffalo.edu/high-throughput-crystallization-center/crystallization-research/. I would recommend the “What’s in a drop?” paper. If there are any homologous proteins that have been crystallized than those conditions can be a good starting guide. There are some proteins that are far more soluble than typical and can have concentrations almost an order of magnitude greater and the occasional on an order of magnitude less. I don’t remember an absolute study on this but I’m sure there must be as I vaguely remember advice that was size related, smaller proteins requiring higher concentration, larger ones less. This may jog someone’s memory to provide the reference. Forgive me for a blatant advertisement, but there is a very successful crystallization screening service at http://getacrystal.org that screens a large array of conditions in a manner to extract this kind of information from them, provides visual, SONICC and UV imaging, and can study the process at multiple temperatures (very useful from a solubility point of view and preserving samples that may be more transient). We hope to implement MARCO (https://marco.ccr.buffalo.edu/) very soon (which has to be added to the reference list – we slipped up there) so you don’t even need to look at all the images. This is a machine vision system we have been developing with many collaborators – it works! (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0198883). As Janet Newman so elegantly said recently, may your New Year Resolutions be high. Best, Eddie Edward Snell Ph.D. Director of the NSF BioXFEL Science and Technology Center President and CEO Hauptman-Woodward Medical Research Institute BioInnovations Chaired Professorship, University at Buffalo, SUNY 700 Ellicott Street, Buffalo, NY 14203-1102 hwi.buffalo.edu Phone: (716) 898 8631 Fax: (716) 898 8660 Skype: eddie.snell Email: esn...@hwi.buffalo.edu<mailto:esn...@hwi.buffalo.edu> Webpage: https://hwi.buffalo.edu/scientist-directory/snell/ [cid:image002.png@01D5C6D4.DD9518C0] Heisenberg was probably here! From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger Rowlett Sent: Wednesday, January 8, 2020 11:29 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: Re: [ccp4bb] Protein concentration for the initial crystallisation trials I usually set a partial screen, maybe 24-48 conditions. If less than half the wells have precipitate, double protein concentration. If most have precipitate, maybe reduce protein or halve concentration of screen reagents. I usually start at 10 mg/mL or so. You can conveniently change protein conc. by manipulating protein/screen volume ratio. __________________ Roger Rowlett On Wed, Jan 8, 2020, 11:16 AM Armando Albert <xalb...@iqfr.csic.es<mailto:xalb...@iqfr.csic.es>> wrote: Dear all, I was wondering how to guess the optimal protein concentration for the initial crystallisation trials. Is there any trick or assay other than the classic PCT from Hampton? Armando ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
