Protein Crystallization Hits

You set up a primary crystallization screen and don't see any crystals at all. You're close to giving up and start thinking about the next construct and another prep. The thing though with protein crystallization is that you may be very close to getting crystals, there's just one thing missing. Since you've got nothing to lose anyways, why not make the best of your crystallization trial and add another crystallization dimension.

1. Rescue clears
lots of clear drops. Dehydrate the drop and well solution by opening the well for a few minutes and then closing again. Concentrating protein and precipitation reagent may be push your crystallization experiment into the crystal nucleation and growth zone. Watch out those salt crystals though!

2. Dissolve precipitate
A primary vapor diffusion crystallization screen does not yield any crystals at all, lots of precipitate. Dissolve the precipitate with 3 to 5 x drop volume of destilled water and let the experiment equilibrate. Some of your precipitate may dissolve and get a new start for nucleation and growth. This time around with less contamination if the contaminants don't dissolve.

3. Seed
Seed with precipitate, with microcrystals, with whatever you have. Sacrifice a well, and mix for instance 1 vol drop with 10 vol well solution and inoculate each remaining drop on the plate with that mixture. Reseal and wait.

4. Change the temperature
You crystallized at 20C? Take the plate out and incubate at room temp - or put it into the fridge. Crystallized in the cold room? Take it out and let it sit at room temp. Any temperature variation has the potential to get you into the sought after nucleation and crystallization zone.

5. Check with a high-magnification microscope
See that shiny precipitate? To get a high magnification view of your drop you may need to take out a portion of the drop and put it onto a microscope slide. Then cover with a glass cover and seal the edges with nail polish to prevent it from drying out. With the highest magnification microscope - use phase contrast if you have it - check out the shape of what may turn out to be microcrystalline material. Count objects without facets but transparent body as a hit and use them as a start for optimization experiments. Or check with #3: use for seeding.

6. Forget about it
Yep, just wait. Ever heard of 'crystal found during lab cleanup' ?. Seriously, over time a lot of things can happen: water diffuses through the plastic, concentrating the contents; those proteases that shouldn't be there munch on your proteins and produce a well-crystallizing proteolytic fragment. Patience is a virtue.

Anybody there with tip7 and 8?