What you should know before starting crystallization
Protein purity: High purity, homogeneity and stability of the protein sample are critical factors for successful crystallization experiments. Your protein should be simply as pure as you can get it! Ideally more than 98%, but at least 90%. Always run SDS-PAGE to make sure that the sample is pure. There are other recommended techniques allowing you to check purity, homogeneity and stability. Dynamic light scattering (DLS) may be used to characterize the polydispersity of the sample (the presence of different oligomeric forms or even aggregates of the protein in solution), and differential scanning fluorometry (DSF) for characterizing the stability of the protein in different buffers and in the presence of different ligands. 1D NMR can be performed to check if a protein is properly folded. Native PAGE, iso-electric focusing gel electrophoresis, and mass spectroscopy on the protein of interest can also be very useful. One should keep in mind that proper and detailed characterization of a protein preparation in crystallography is much more important than in biochemical experiments, where often partial purity and the activity of the protein are taken as sufficient criteria for the quality of the preparation.
Sample concentration: Before beginning crystallization trials, the sample needs to be concentrated and transferred to dilute buffer containing little or no salt if the protein is happy under these conditions. This can easily be achieved using centrifugal concentrators. Buffer can be also exchanged during size exclusion chromatography step. Protein concentration of 1-25 mg/ml is recommended. It is always case dependent, 10-15 mg/ml is typical, but crystals have been grown using 0.5‑100 mg/ml. Eukaryotic proteins tend to be less soluble that bacterial proteins. If you know that your protein aggregates at 10 mg/ml, then there is no point to concentrate it so much.
Amount of the sample: To perform a single crystallization screen (96 different conditions using the smallest available drop volume) you will need about 15 microliters of the sample. About 10 microliters will be used up and another few microliters must be present to ensure that the needles of the mosquito robot suck up protein solution and not air. To set up all available screens (12) bring at least 150‑200 microliters of your precious protein of the proper concentration.
Sample buffer: If your protein is stable, ideally use water so it will not interfere with conditions used for crystallization trials. However, most proteins have poor solubility and stability in water therefore weak (10-50 mM) buffers (TRIS or HEPES is the best choice) with some amounts of salt (10-100 mM) can be used. If possible avoid high salts! Do also avoid phosphate and ammonium sulphate as these can cause problems! However, if your protein requires such suboptimal conditions it is more important to have a stable protein sample than an optimal buffer condition.
Storage of Protein: Not all proteins tolerate freezing at -20°C. Most proteins can be kept at 4°C or -80°C, but the activity and stability must be checked for each new protein. Freezing and re-thawing of the sample should be avoided, so store the protein in aliquots. The best is to freeze the protein (we usually prefer without any glycerol) directly in liquid nitrogen and store it at -80°C. As a general rule, it is better to store proteins concentrated than diluted. Handle the protein solution gently. Avoid foam. Do not vortex or shake it. If your protein stock solution is stored at a temperature different from the one at which crystallization trials will be set up, a good practice is to equilibrate your aliquot before to the target temperature.
- If particles or amorphous matter is present in the sample, centrifugation or micro-filtration is advisable. Centrifugation (15-30 min at max speed) is also recommended to get rid of air bubbles.
- If possible make size-exclusion chromatography your final step of the purification. Use the final buffer for crystallization already during the last chromatography experiment. Dialysis or exchanging the final buffer using centrifugal concentrator is possible but not recommended.
- Select only the best fractions from the size-exclusion chromatography for crystallization.