Initial crystallization conditions from the Crystal Former can be optimized in several ways. In general, we do not recommend attempting to calculate the concentration of the reagent at a specific time and position in the channel where your crystal appeared. Rather, we suggest the following simple approaches:
 
  • Vary the ratio of protein volume to precipitant volume. The resultant change in the crystallization gradient has direct impact on the nucleation rate, and can thus significantly alter the crystal size.
  • Set up a coarse grid screen around the initial condition in the Crystal Former 16-chnnel chips (original or scale-up) or a few columns of the CF-HT2 plate (cut the sealing film to size. Depending on the amount/type of crystals and amount of precipitation in the original channel, the grid screen can be shifted towards higher or lower concentrations of precipitant(s).
  • Crystal Former hits can often (but not always) be reproduced and optimized in vapor diffusion format. Use a grid screen around the initial condition to optimize. The initial condition concentrations may be changed significantly during optimization process and it is especially important to test high concentrations of the precipitant when moving into vapor diffusion.

 

 

Conditions from the PurePEGs screens

Thr PurePEG cocktail consists of 4.5% mix of each of the following PEGS: 300, 400, 1000, 4000, 8000. You can purchase the individual, USP grade PEG components directly from Microlytic. The 50% PEG cocktail is also available for purchase.

 

You can optimize your crystals by setting up a grid screen around the initial condition, varying the concentration of the PurePEG cocktail along one axis, like you would with any other reagent. Since the concentration gradient is explored in each capillary, the grid screen does not need to be as fine as the one typically devised for hit optimization in VD. 

 

However, optimization of crystallization hits from the PurePEG screen is not necessarily contingent on use of the PEG cocktail.  Users may utilize small, medium and large MW PEGs to do three parallel grid screens around the initial hit. For many PEG-containing conditions, the identity of the PEG is not absolute for crystal reproducibility. PEG compounds can often be substituted for one another, though a wider range of PEG concentrations should be explored. 

 

The major consideration for the reproducibility of crystal hit from the PurePEG screen is the PEG purity. Most PEG compounds utilized for crystallization have not traditionally been USP grade. As such, the presence of contaminants in low PEG grades may hinder the reproducibility of crystals.