4Ī range of conventional methods can be used to determine the isoelectric point of proteins, including isoelectric precipitation, 5 techniques based on ion-exchange adsorption, 6–8 zeta potential measurements, 9–11 capillary electrophoresis 12 or a recently developed nanoparticle-based approach. 1 Knowledge of the value of pI of a protein is particularly useful for separation 2 and purification, 3 and for the characterisation of key physicochemical properties, such as surface charge and solubility, which are typically lower at values of the pH in the vicinity of the pI. 1 Introduction The isoelectric point (pI) is the value of the pH of a solution at which amphoteric molecules, such as proteins, have a vanishing net charge and hence no effective electrophoretic mobility. The ability to conduct measurements in free solution thus provides the basis for the rapid determination of isoelectric points of proteins under a wide variety of solution conditions and in small volumes. To demonstrate the general approachability of this platform, we have measured the isoelectric points of representative set of seven proteins, bovine serum albumin, β-lactoglobulin, ribonuclease A, ovalbumin, human transferrin, ubiquitin and myoglobin in microlitre sample volumes. In particular, in this approach, the pH of the electrolyte solution is modulated in time rather than in space, as in the case for conventional determinations of the isoelectric point. Here, we introduce a gradient-free approach, exploiting a microfluidic platform which allows us to perform rapid pH change on chip and probe the electrophoretic mobility of species in a controlled field. The majority of conventional methods for the determination of the isoelectric point of a molecule rely on the use of spatial gradients in pH, although significant practical challenges are associated with such techniques, notably the difficulty in generating a stable and well controlled pH gradient. The isoelectric point (pI) of a protein is a key characteristic that influences its overall electrostatic behaviour.
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