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Sticky crystals: Difference between revisions
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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. | 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 | COAT THE CRYSTALLIZATION SURFACE WITH A THIN LAYER OF GREASE | ||
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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. | 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. | One may opt for Vaseline or petroleum jelly rather than silicone-based grease. | ||
COAT THE CRYSTALLIZATION SURFACE WITH SILICON | COAT THE CRYSTALLIZATION SURFACE WITH SILICON | ||
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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 eg. Repelcote. | 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 eg. Repelcote. | ||
CHANGE CRYSTALLIZATION PLATE | CHANGE CRYSTALLIZATION PLATE | ||
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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 | |||
CRYSTAL BOWLING | CRYSTAL BOWLING | ||
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If you have good and bad crystals in the same drop, | 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. | ||
TRY CRYSTALLIZATION WITH AGAROSE AS AN ADDITIVE | |||
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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: | Reference: | ||
Biertmpfel, C.; Basquin, J.; Suck, D. & Sauter, C. | Biertmpfel, C.; Basquin, J.; Suck, D. & Sauter, C. | ||
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PMID: 12351881 | PMID: 12351881 | ||
FLOATING DROP CRYSTALLIZATION METHOD | |||
'FLOATING-DROP' CRYSTALLIZATION METHOD | |||
References | ------------------------------------ | ||
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 | 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 | ||
MICROSEEDING | MICROSEEDING | ||
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This method should allow better control of nucleation events in case the | |||
DRY-ICE trick | DRY-ICE trick | ||
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GROW THE CRYSTALS ON A MICROTOOL MESH | 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. | 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. | ||