I tried Hampton Screen I & II with my protein and have no crystals. What can I do?Edit
- If most of your drops have precipitate, halve the concentration of the screening reagents, and try again. If setting 500 uL wells, simply pipet 250 uL of water and 250 uL of screening reagent in each well, mixing thoroughly. Standard screens, including Hampton I and II, are often too concentrated for efficient nucleation and crystallization for some proteins. If you still have precipitate in most of the drops after halving the concentration, halve the concentrations again.
- Vary protein concentration. This is rather obvious suggestion when all your drops are clear or all have precipitate, in which case your overall concentration is too low (too high). More elaborate approach is to modify concentrations based on the results of your first screen, i.e. double it for the conditions which produced clear drops and cut it in half for those which produced precipiate. To avoid concentrating/diluting protein, you can simply try to mix your original protein stock with reservoir solutions in different proportions. For instance, mixing 2ul of protein with 1ul of reservoir solution is (to some degree) equivalent of doubling protein concentration.
- Set your screens at different temperature. 25 and 4 degrees are the most popular options. If you completely lost hope for your original screens, just transfer them to the cold room. Protein solubility will change and you may get crystals.
- Add a purification step. Purity can be critical to protein crystallization and the purity judged from SDS-PAGE or even gel filtration can be misleading.
- There are other screens. While it is proably true that when proteins do crystalize, they in many cases produce hits from Hampton screens I or II, we are dealing here with exception to that rule. Lists of screens are available from manufacturer websites:
- Modify your protein.
Reducing the mosaicityEdit
Things to try concerning crystallization - see also Improving crystal quality.
For cryo question see cryo and Data collection: Tips and Tricks.
- change precipitant, pH, temperature; try additives
- try co-crystallization with glycerol (2%, 5% or 10%).
- incubate your crystal longer in your cryo/stabilization solution.
- optimize the cryo conditions
- anneal (freeze/thaw) the crystal - DISCLAIMER: your mosaicity can go up as well as down with annealing
- the mosaicity may be anisotropic, so try to orient the crystal in the loop such that you can shoot along a different axis
- look at Elspeth Garman's papers, and/or check her talks at RapiData workshops (anybody with a link??)