Sticky crystals

Crystal harvesting out of native crystallization drops (hanging- and/or sitting-drop geometries) can sometimes be severely hampered by the propensity of crystals to stick to their crystallization surface. Here are some tips on how to approach the problem:

OPERATE ON THE PLASTIC RATHER THAN ON THE CRYSTAL!

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-I dig the microtool into the plastic as close as I can get to the crystal without touching the crystal. Usually a small deformation of the plastic causes the crystal to pop off intact. -I have had similar problems with crystals sticking to the plastic of the crystallisation plates (96-well Grenier plates). I use an acupuncture needle and dig into the plastic right next to the crystal, directing the needle below the crystal. By digging and twisting, you can generate little movements across the plastic which can be enough to free the crystal. If you slip, you end up playing golf with your crystals, but with a steady hand, it has worked well for me, though I guess it might be quite dependant on the strength of the plastic of the plate. Worth trying though if you have crystals. -Take a sturdy needle (like one of the microneedles from a Hampton kit or a very thin syringe needle) and (while observing the whole thing under a scope) stick the needle into the plastic a bit away from the crystal. Push hard. If you’re using polarizers, you may be abe to visualize the stress forces in the plastic by the shifting of the colors. The basic idea here is to stress the plastic under the crystal w/o touching the crystal in any way. In my case the bloody things just popped off. Sometimes you have to push quite hard, and to wiggle the needle a bit – and beware, they can slip and ruin your drop. But this really worked quite well!

COAT THE CRYSTALLIZATION SURFACE WITH A THIN LAYER OF GREASE

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-Additionally, once I realized this was going to be a long term problem, I started coating the sitting drop depressions with a thin layer of vacuum grease. The crystals just slid right off the grease and I never saw any changes in the diffraction data to suggest the grease was giving me issues. -You only need a very thin layer of the grease (i.e. keep wiping until its almost completely gone) and it usually has no affect on the crystallization. -Another approach is to grease the wells of the plate. If you then gently warm and melt the grease (use Vaseline or other petroleum jelly rather than silicone grease) it will set almost clear. This is sometimes used with microbatch.

COAT THE CRYSTALLIZATION SURFACE WITH SILICON

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You can try various siliconizing fluids. Hampton used to sell one called Aquasil which you mix up in water. This will not melt the plastic as eg. Repelcote will.

CHANGE CRYSTALLIZATION PLATE

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You can also try plates made from COC (cyclic olefins), such as the "UVP" plates made by SwissCi (sold by lots of companies including us). They are less sticky than polystyrene plates. COC is halfway between polystyrene and polypropylene. Polypropylene is even less sticky than COC but is not rigid, therefore harder to dispense to automatically. You can get plates made of "clarified polypropylene" from Emerald, and you can also get polypropylene "bridges" that you place in Linbro wells. I think Hampton still sells them.

CRYSTAL BOWLING

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If you have good and bad crystals in the same drop, I've had success pushing a crummy crystal into a good crystal and having it release that way.

CRYSTALLIZATION WITH AGAROSE AS AN ADDITIVE

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Crystals form inside the very soft gel and they are hold in place by this meshwork. So, they are mechanically protected and do not fall down onto the bottom of the sitting-drop well. A final concentration of 0.1-0.2 % (w/v) agarose is sufficient. When you harvest a crystal cut generously around it with a microtool, pick it up (e.g. using a nylon loop) and do not mind if some agarose comes with it. Reference: Biertmpfel, C.; Basquin, J.; Suck, D. & Sauter, C. Crystallization of biological macromolecules using agarose gel. Acta Crystallogr D Biol Crystallogr, 2002, 58, 1657-9 PMID: 12351881

FLOATING DROP CRYSTALLIZATION METHOD

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References 1. Application of a two-liquid system to sitting-drop vapour-diffusion protein crystallization. Adachi, H. et al, Acta Cryst. (2003) D59, 194-196 2. Promotion of large protein crystal growth with stirring solution. Adachi, H. et al. Jpn. J. Appl. Phys. Vol. 41 (2002) pp.1025-1027 3. Two-liquid hanging-drop vapour-diffusion technique of protein crystallization. Hiroaki Adachi et al. Japanese Journal of Applied Physics. Vol. 43, No. 1A/B, 2004, pp.L79-L81. Here is a web link to the fluid on the Hampton Research web site. http://hamptonresearch.com/product_detail.aspx?cid=4&sid=185&pid=32 HR2-797 100% Fluorinert FC-70 Fluid 100 ml

MICROSEEDING

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DRY-ICE trick

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Put a small piece of dry ice on the opposite side of the plastic from the crystal. Perhaps the difference in thermal expansive coefficient will let the crystal(s) break away. Don't overdo it though. This is a trick that Gary Gilliland taught me.

‘IN-PLATE’ DIFFRACTION EXPERIMENTS

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-i hope i understood correctly, but basically leave the crystal in-situ and put the whole ensemble in an x-ray beam. somehow. you may see plastic scatter. never tried it, all theoretical. perhaps custom-cut a tray so you can break the well away when the xtal grows. or put something down there to grow on, then pick it out... would love to know if any of that works! -You might want to contact Luc Ferrer from ESRF in Grenoble or read his publications. I know they were developping robotisation for in plate shooting, but you probably will need to set up new trials in a particular type of plate. -You can collect data on your crystal still in the drop, on our beamline (FIP-BM30A at the ESRF) if you are interested. Provided space group is not P1.... We do that routinely. MITEGEN MICROTOOLS

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The MiTeGen microtools kit: http://mitegen.com/products/microtools/microtools_kit1.shtml comes with a "MicroSaw", which is a 10-micron thick kapton saw that is intended for this purpose. That is, you don't pry the crystal off the surface, but rather rest this saw against the surface, bring it over to the edge of the stuck crystal and then work it back and forth until you have cut underneath the crystal. PIPETTING techniques I had a similar story like yours.Then I added a drop of 10ul simulated mother liquot which contains much higher concentrations of all components in the normal mother liquot. Sometimes, the crystals attached to the plastic would float to the surface. If not, take another 10ul, but blew it to the bottom plastic with a pippetman back and forth, and some crystals would also leave the plastic(But you have to be very careful to do this.)

GROW THE CRYSTALS ON A MICROTOOL MESH

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SONICATION

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For my PhD I once sonicated crystals off a glass surface; diffraction was fine; apparently this was the standard approach for papain when Jan Drenth solved it long ago.