Hi,
It sometimes comes down to manual dexterity - do you have a friend with 'surgical hands'? It's sometimes possible to simply cut a little 'window' in the skin and to harvest the crystal, skin and all - usually the skin is not significantly ordered and thus does not pose a problem for data collection. In many cases the skin formation may be inhibited by addition of 'insane' quantities of reducing agents (e.g. 2% BME or 25 mM DTT). In other cases this problem can be solved by adding small amounts of glycerol or ethylene glycol (or less commonly - isopropanol) to the condition. I have had one success with detergents, however in several other cases the addition of detergents made the skin even thicker/nastier/stickier. Sometimes protein crystals attached to skin can be liberated during cryoprotection by placing a small drop of cryo-laden reservoir solution directly over the attached crystals (from the outside of the crystallization drop!). I am not sure how this works (when it does, that is) but I guess that the gradient of cryo-agent can cause the skin layer to shrink or expand slightly, which gently releases the crystals. Once you have tried this - don't forget to gently poke the crystals through the skin with an e.g. a cat whisker - sometimes the crystals don't just fall off by themselves, but instead become amenable to be detached by gentle mechanical means. If all else fails, you could try trypsinizing the skin away before harvesting crystals. It's not an easy or fun process and its success depends on the compatibility of your crystallization condition with trypsin. The way I do it (on a couple of occasions when this was really necessary) is to gently place a very small drop of trypsin-loaded reservoir condition directly over the crystal(s) without disrupting the skin. Then I place the cover slip back and make sure that the seal is tight (it can take minutes or hours for the trypsin to work, can't guess in advance!). Then I wait and watch - typically when trypsin has done its job you will see the two drops suddenly merge, and the skin will have a nice hole through it. Obviously, once the skin over your crystals is gone, it's a good idea to freeze the liberated crystals quickly! Artem _____ From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of riya doreen Sent: Sunday, December 28, 2008 8:24 PM To: [email protected] Subject: [ccp4bb] crystals from skin Hello All, I am trying to optimize protein crystals that grow from skin. All efforts to separate crystals from skin have failed since it is fairly thick. Crystallization under oil eliminates skin but the resultant crystals are very thin and small needles. I would appreciate any suggestions on how this problem can be eliminated. Thanks
