The pH of a solution is an important parameter in crystallization that needs to be controlled in order to ensure success. The actual pH of the crystallization droplet is determined by the combined contribution of the buffers in the screening and protein solutions, although the contribution of the latter to the pH is often ignored. In this study, the effects of the buffer and protein solution pH values on the results of screening are systematically investigated. It was found that these parameters significantly affected the results and thus the following strategy for the selection of appropriate pH values is proposed: (i) when screening with only one protein solution, the pH should be as low, as high or as divergent from the pI as possible for a basic, acidic or neutral protein, respectively, within its stable pH range; (ii) when screening with two protein solutions, the pH values should be well separated from one another; and (iii) when multiple pH values are utilized, an even distribution of pH values is the best approach to increase the success rate of crystallization.
Keywords: crystallization screening; pH; protein crystallization.