The main challenges in screen making are accuracy and reproducibility. For labs with high volumes of experiments, the time to set up screens can also become a limiting factor, but without accuracy and reproducibility, protein crystallization becomes a hit or miss affair.
Reproducibility affects our ability to optimize a screen around a hit. Ideally, you would like the new design to be centered around your hit. If you cannot reproduce the conditions of your prior design, an “optimized” screen might be so far off that no crystallization event occurs in your next experiment at all.
Of course there is no reproducibility without accuracy, but inaccurate screens cause problems even when reproducibility is of no concern. Even easy to crystallize proteins require careful control over the chemicals dispensed into the screen and lack of control over the dispensing process can lead to failures further down the pipeline.
Another issue that can affect accuracy and reproducibility is cross contamination, or the accidental mixing of chemicals as there are being dispensed into the screen. Cross contamination is best controlled by strictly separating all chemicals during the dispensing process. Another option is to purge and wash the dispensing pathways before dispensing a new chemical, but this can cause issues with poorly soluble chemicals and can create waste.
Accuracy of screen makers should be tested for the types of chemicals relevant for protein crystallization. Shown here is a CV (coefficient of variation) test for 50% PEG (above) and pH test (below).
The accuracy achieved by automated screen makers is best tested via coefficients of variation – “CVs”. They are a measure of how much an automatically dispensed volume of chemical deviates from the ideal volume specified by the controlling software. The accuracy is best benchmarked against serial dilution experiments, the most accurate (but unfortunately for our purposes not practical) dispensing method available.
When testing for accuracy, it is important to test CVs for all viscosities needed to make a screen and particular attention should be given to the most viscous materials one will have to dispense since they typically will show the highest variabilities.
Another important aspect of fine screen making is pH control. An automated fine screen maker should give you the same level of pH control as for dispensing volumes. This can be accomplished via interpolation algorithms a la Hasselbacher or by lookup from actual titration curves. Ideally the controlling software should be able to support both scenarios.
More posts on screen making.
Click here to learn more about screen making at Rigaku.com.
