Filtration: simple sample pre-treatment for protein crystallization experiments
Posted by Peter Nollert on Tue, Jun 30, 2009 @ 12:22 PM
One of Naomi Chayen's best advice on crystallization optimization she's given me was "always filter your sample for optimization". In more detail: she advocates filtering protein samples with filters of increasingly smaller mesh size (such as 0.22 micron, 0.1 micron and 300 kDa MWCO). The filtered solutions are then each used to set up optimization crystallization trials. That's simple to do when sample quantity is ample since you'll lose ca.5-10 ul in filter dead volume for each filtration run. Naomi has described the procedure in several of the talks that I had the pleasure to attend. And more recently she's condensed her filtration results and published it in a laboratory note in the
Journal of Applied Crystallography (2009, 42) fittingly titled: "Rigorous filtration for protein crystallization". I like the word 'rigorous' in this context - sounds like: forcing out the rubbish. She's making the point that "Filtration is relevant to all methods of crystallization for both screening and optimization".
The thinking here is of course to clear up the sample by removing fines from chromatography resin, dust, aggregated protein and to reduce the number of nuclei, hence suppress heterogenous nucleation. The latter of course comes in handy when the goal is to convert showers of crystals into one single well diffracting crystal. The table she shows is impressive. The number (reduced from >1000 to 0), size (10 um to 600 um) of crystals grown from aliquots of the same protein solution under otherwise identical conditions depended on filter mesh size used (she compared unfiltered, 0.22 um, 0.1um, 0.1 um and 300 kDa MWCO).
The advantages of filtration are clear (sic!): growing better crystals right away and creating solutions that are ready for seeding. The biggest advantage of rigorous filtering in my mind however is that this is an additional tool to optimize without having to change the crystallization conditions.
Big thanks to Naomi!
Peter
