Crystaliztion condition screening
The quality of the protein is critical for a successful crystal formation. I generally double purify the protein with two gel filtration columns of different brand, and collect only the central 75% of the peak. Also try to use low salt concentration buffer to store the protein. Phosphate buffer may form artificial salt crystals. In my previous experiments I have screened many conditions for growing complex crystals of uPA protease and bicyclic peptides. It is essential to completely block the activity of the protease with the inhibitor to avoid auto degradation.
In the robotic screening stage, I have varied the concentration of protein (15mg/mL, 10mg/mL, 6mg/mL) and also the concentration of inhibitors (1mM for potent peptides, 2~3mM for peptides with moderate inhibitory activity and 3mM for small molecules). The commercially available screening kits that I like are Clear Strategy Screen I HT, Clear Strategy Screen II HT (MOLECULAR DIMENSIONS) and Crystal screen HT (HAMPTON). The robot I used is mosquito crystal by mixing 0.075uL reservoir solution and 0.075uL protein+peptide mixture (preincubate in ice for 0.5h). In the 96-well sitting drop plate, there are three drop spot in each well to allow screening three protein conditions in parallels. When crystals are discovered in the wide screen, I generally verify the conditions on 24-well plate by mixing 1.25uL Reservoir solution with 1.25uL protein solution.
To optimize crystal growing conditions, I generally perform manual screening in 24-well plate. At this stage, I always prepare the buffer stocks and filter them. Commercially available buffers are also available. I prefer to vary all the parameters of the positive screening from previous step. In my case, pH is critically important. The reason I thought is because peptides can carry different number of TFA, which can change the pH of the protein solution much. To slow down the growth speed of crystals, 5% glycerol can be added to the reservoir solution.
After fishing the crystal, I cryo-protect the crystal in 1uL 50% glycerol+ 1uLreservoir solution for 10s. Then flash freeze it. I prefer not to cut crystals, because it was hard for me to avoid cracks in the crystal.