Hi Nikolaus I completely agree with Claude's comments. Microseeding is the first thing to try. You would like to find conditions where you *don't *get crystals without seeding, but you *do *get crystals with seeding. Then, just by diluting the seed stock, you can control nucleation. Look at D'Arcy and Obmolova's papers, links below (second time!).
I also agree that crystallization in agarose can work well - if seeding doesn't solve your problem. The only person that I know who tried containerless crystallization ended up with more crystals rather than fewer. Actually your case is not very unusual - when you scale up you tend to get *more *crystals. The reason is that the surface-area-to-volume ratio is greater for smaller drops. This means that you lose a greater proportion of the protein on the plastic or glass surface of your plate, and also on the air-drop interface. Therefore you should dilute the protein and/or the precipitant when you scale up. Good luck, Patrick http://scripts.iucr.org/cgi-bin/paper?S2053230X14015507 https://scripts.iucr.org/cgi-bin/paper?nj5193 On Mon, Sep 9, 2019 at 4:45 PM Claude Sauter <c.sau...@ibmc-cnrs.unistra.fr> wrote: > Le 09/09/2019 à 17:22, Nikolas a écrit : > > Dear crystalgrowers, > > I am currently working with a protein that appeared to be friendly but > turned out it was not the case. > I found myself to face -in the scale up- the opposite of the usual problem > of nucleation (I really love how this topic finds new ways to make fun of > me). In 24-well plates, hanging-drop, for the same condition but in > different drops I found few big but intergrown crystals and/or a full with > microcrystals. Sometimes also in the same well, when having more drops. > I already decreased the concentration to less than 4mg/mL, made small > adjustments in the optimizations - both with apo and ligand samples, used > Al's oil. > > I have read about the "containerless crystallization" but since I cannot > obtain the sample myself I would like to know if there are any experiences > and/or if there are suggestions for solving this problem. > > Many thanks! > > Best regards, > Nikolas > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > Dear Nikolas, > > since you have some crystal stock, I would definitely try seeding to > better control nucleation events in your drops. Then, instead of using the > containerless approach which requires two types of oils to prepare floating > drops, I suggest the crystallization in agarose gel. Easy to perform, it > favors the 3D growth of well separated crystals in ideal convection-less > conditions. In addition, the gel provides a physical protection of your > crystals during handling, mounting and cryocooling. For more details, see > "Crystal > growth of proteins, nucleic acids, and viruses in gels. Lorber et al. Prog. > Biophys. Mol. Biol. (2009), 101: 13-25." > > Happy crystallization! > > Claude > > -- > Dr Claude Sauter > Institut de Biologie Moléculaire et Cellulaire (IBMC-ARN-CNRS) > Biologie des ARNt et pathogénicité tel +33 (0)388 417 102 > 2 allée Conrad Roentgen fax +33 (0)388 602 218 > F-67084 Strasbourg - France http://cj.sauter.free.fr/xtal > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > -- patr...@douglas.co.uk Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
