11 things I wish I had known about protein crystallization optimization when I set up my first optimization trays
Posted by Peter Nollert on Tue, Jun 16, 2009 @ 10:27 AM
- No need to get excited about crystals in the drop if there are salt crystals the 'mother liquor'.
- Those clear but irregularly shaped objects without facets that look like glass shards that show up in some hanging drops are indeed small pieces of glass that broke off when the cover slides were made.
- Those soft-looking, clear objects on the bottom of the sitting drop wells are not 'pre-crystalline material', they're plastic tray manufacturing artefacts.
- Shiny precipitate is a good start for crystal growth optimization.
- The color that's associated with very small crystals is not the chromophore of the protein but stems from chromatic aberration of the microscope lens.
- Better save a small quantity of shock-frozen protein solution and use as a positive control in follow-up optimization trials than making another prep.
- Take an image of the first crystals right when I see them for the first time. Take any image - use my phone if necessary. Otherwise nobody will believe that there were indeed crystals at 16C that 'melted' after tansferring the crystallization tray to the 21C lab with the high-resolution digital camera mounted on the microscope.
- It doesn't hurt to take crystals out of the drop, solubilize them in SDS and run them out on a gel. If the main band does not run with the target protein, something's fishy - or something interesting is going on.
- The 'flittery precipitate' that formed in the protein solution right before setting up the crystallization trial may be showers of microcrystals. Better check a small sample with a high-magnification microscope.
- Do label the crystallization tray, not the cover (of the batch-under-oil crystallization experiment).
- I will forget the meaning of "optimized hit condition #4" when it's paper writing time.
