Difference between revisions of "Sticky crystals"

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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:
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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 (sometimes very tightly) to their crystallization surface. Here are some tips on how to approach the problem:
  
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OPERATE ON THE PLASTIC RATHER THAN ON THE CRYSTAL!
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Take a sturdy needle (like one of the microneedles from a Hampton kit, or a very thin syringe needle, or an accupuncture needle) and 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 is to stress the plastic under the crystal without touching the crystal in any way. By digging and twisting, one can generate little movements across the plastic which can be enough to free the crystal. Sometimes you have to push quite hard, and to wiggle the needle a bit. Beware, however, as the needle can slip and ruin the crystallization drop.
  
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!
 
  
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COAT THE CRYSTALLIZATION SURFACE WITH A THIN LAYER OF GREASE
 
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.
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If working with the 'sitting-drop' geometry, one can coat the sitting drop depressions with a thin layer of vacuum grease. You only need a very thin layer of the grease (i.e. keep wiping off with a KimWipe until the grease is almost completely gone). Upon crystal harvesting, the crystals will have the tendency to slide right off the grease.
-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.
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One may opt for Vaseline or petroleum jelly rather than silicone-based grease.  
-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.
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COAT THE CRYSTALLIZATION SURFACE WITH SILICON
 
COAT THE CRYSTALLIZATION SURFACE WITH SILICON
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You can try various siliconizing fluidsHampton used to sell one called Aquasil which you mix up in waterThis will not melt the plastic as eg. Repelcote will.
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You can try various siliconizing fluids such as AquaSil (Hampton Research)Such a product will not melt the plastic of the crystallization plate as opposed to e.g. Repelcote.
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CHANGE CRYSTALLIZATION PLATE
 
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.
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One may want to try plates made from COC (cyclic olefins), such as the "UVP" plates made by SwissCi. 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 not recommended for crystallization robots.  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.
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.
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CRYSTAL BOWLING
 
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.
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If you have good and bad crystals in the same drop, one may try to push crummy crystals into a good crystal, thus creating the necessary leverage to release the useful crystals from their growth position.  
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CRYSTALLIZATION WITH AGAROSE AS AN ADDITIVE
 
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.
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Crystals grown in the presence of 0.1-0.2 % (w/v) agarose will grow inside the soft agarose gel.
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.
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Therefore, they are mechanically protected and will not settle to the bottom of the sitting-drop well. 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.
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Reference:
 
Reference:
 
Biertmpfel, C.; Basquin, J.; Suck, D. & Sauter, C.
 
Biertmpfel, C.; Basquin, J.; Suck, D. & Sauter, C.
Line 37: Line 44:
 
PMID: 12351881
 
PMID: 12351881
  
FLOATING DROP CRYSTALLIZATION METHOD
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References
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'FLOATING-DROP' CRYSTALLIZATION METHOD
1.      Application of a two-liquid system to sitting-drop vapour-diffusion protein crystallization. Adachi, H. et al, Acta Cryst. (2003) D59, 194-196
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2.      Promotion of large protein crystal growth with stirring solution. Adachi, H. et al. Jpn. J. Appl. Phys. Vol. 41 (2002) pp.1025-1027
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References:
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.
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-Application of a two-liquid system to sitting-drop vapour-diffusion protein crystallization. -Adachi, H. et al, Acta Cryst. (2003) D59, 194-196
Here is a web link to the fluid on the Hampton Research web site.
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Promotion of large protein crystal growth with stirring solution. Adachi, H. et al. Jpn. J. Appl. Phys. Vol. 41 (2002) pp.1025-1027
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-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.
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THe necessary reagents for applying this method can be found at:
 
http://hamptonresearch.com/product_detail.aspx?cid=4&sid=185&pid=32
 
http://hamptonresearch.com/product_detail.aspx?cid=4&sid=185&pid=32
 
HR2-797        100% Fluorinert FC-70 Fluid    100 ml
 
HR2-797        100% Fluorinert FC-70 Fluid    100 ml
  
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MICROSEEDING   
 
MICROSEEDING   
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This method should allow better control of nucleation events in case the crystallization surface.
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DRY-ICE trick
 
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.
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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.   
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‘IN-PLATE’ DIFFRACTION EXPERIMENTS
 
‘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.
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Beamline FIP-BM30A at the ESRF (contact Jean-Luc Ferrer) has a setup that allows crystal testing, even data collection, while the crystal remains in the crystallization drop (see movie at [http://www.natx-ray.com/products/G-Rob_2D_movie.html]). A lab system is commercialized by NatX-ray [http://www.natx-ray.com/products/G-Rob_2D.html].
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!
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-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.
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-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.
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MITEGEN MICROTOOLS
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MiTeGen MICROTOOLS
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The MiTeGen microtools kit:
 
The MiTeGen microtools kit:
 
http://mitegen.com/products/microtools/microtools_kit1.shtml
 
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.
 
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.
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PIPETTING techniques
 
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.)
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Add a drop of 10ul mother liquor to the crystallization drop. Sometimes, the crystals attached to the plastic would float to the  surface. If not, take another 10ul and pipet it in and out the drop as close as possible to the bottom.  This might dislodge additional crystals from the plastic.  
  
GROW THE CRYSTALS ON A MICROTOOL MESH
 
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SONICATION
 
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.
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One may want to sonicate crystals off its crystallization surface; apparently this was the standard approach for papain when Jan Drenth determined the structure a long time ago.

Latest revision as of 12:23, 4 February 2009

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 (sometimes very tightly) to their crystallization surface. Here are some tips on how to approach the problem:


OPERATE ON THE PLASTIC RATHER THAN ON THE CRYSTAL!


Take a sturdy needle (like one of the microneedles from a Hampton kit, or a very thin syringe needle, or an accupuncture needle) and 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 is to stress the plastic under the crystal without touching the crystal in any way. By digging and twisting, one can generate little movements across the plastic which can be enough to free the crystal. Sometimes you have to push quite hard, and to wiggle the needle a bit. Beware, however, as the needle can slip and ruin the crystallization drop.



COAT THE CRYSTALLIZATION SURFACE WITH A THIN LAYER OF GREASE


If working with the 'sitting-drop' geometry, one can coat the sitting drop depressions with a thin layer of vacuum grease. You only need a very thin layer of the grease (i.e. keep wiping off with a KimWipe until the grease is almost completely gone). Upon crystal harvesting, the crystals will have the tendency to slide right off the grease. One may opt for Vaseline or petroleum jelly rather than silicone-based grease.



COAT THE CRYSTALLIZATION SURFACE WITH SILICON


You can try various siliconizing fluids such as AquaSil (Hampton Research). Such a product will not melt the plastic of the crystallization plate as opposed to e.g. Repelcote.



CHANGE CRYSTALLIZATION PLATE


One may want to try plates made from COC (cyclic olefins), such as the "UVP" plates made by SwissCi. 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 not recommended for crystallization robots. 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


If you have good and bad crystals in the same drop, one may try to push crummy crystals into a good crystal, thus creating the necessary leverage to release the useful crystals from their growth position.



CRYSTALLIZATION WITH AGAROSE AS AN ADDITIVE


Crystals grown in the presence of 0.1-0.2 % (w/v) agarose will grow inside the soft agarose gel. Therefore, they are mechanically protected and will not settle to the bottom of the sitting-drop well. 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


References: -Application of a two-liquid system to sitting-drop vapour-diffusion protein crystallization. -Adachi, H. et al, Acta Cryst. (2003) D59, 194-196 Promotion of large protein crystal growth with stirring solution. Adachi, H. et al. Jpn. J. Appl. Phys. Vol. 41 (2002) pp.1025-1027 -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.

THe necessary reagents for applying this method can be found at: http://hamptonresearch.com/product_detail.aspx?cid=4&sid=185&pid=32 HR2-797 100% Fluorinert FC-70 Fluid 100 ml



MICROSEEDING


This method should allow better control of nucleation events in case the crystallization surface.



DRY-ICE trick


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.



‘IN-PLATE’ DIFFRACTION EXPERIMENTS


Beamline FIP-BM30A at the ESRF (contact Jean-Luc Ferrer) has a setup that allows crystal testing, even data collection, while the crystal remains in the crystallization drop (see movie at [1]). A lab system is commercialized by NatX-ray [2].



MiTeGen MICROTOOLS


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


Add a drop of 10ul mother liquor to the crystallization drop. Sometimes, the crystals attached to the plastic would float to the surface. If not, take another 10ul and pipet it in and out the drop as close as possible to the bottom. This might dislodge additional crystals from the plastic.



SONICATION


One may want to sonicate crystals off its crystallization surface; apparently this was the standard approach for papain when Jan Drenth determined the structure a long time ago.