3/30/2023 0 Comments Isoelectric point measure![]() 16,31 Another potential drawback of CAs-gradients is the cathodic drift that can be due to the electromigration of CAs, electrolyte diffusion or electroosmosis, 30,32 and, moreover, the CAs approach is most reliable typically for samples under low salt concentration conditions. ![]() 31 Moreover, CAs and often markers can also interact with the protein under investigation and thus affect its pI. These compounds undergo a differential drift in an applied electric field and form a gradient, 30 that is nonlinear. 28 A significant improvement in this context is the invention of a “natural” pH gradient, 29 generated by the simultaneous use of several carrier ampholytes (CAs), amphoteric compounds with pI values close to each other. 27 IEF devices with spatial pH gradients have been created simply by mixing acidic and basic buffers, but this approach can be challenging to implement, because of difficulties in ensuring the stability of the gradient. 26 The advantages offered by μFFIEF are rapid focusing of protein mixtures and protein complexes, accurate control of laminar flow and negligible Joule heating, although the quantitative control of maintenance and characteristic of spatial pH gradients remains a limitation. 19–22 One currently used micro-scale approach for determining pI is microfluidic Free-Flow Isoelectric Focusing (μFFIEF), 23–25 which is based on the same principle as IEF with a pH gradient in the direction perpendicular to the advective flow in a microchannel. Microfluidic platforms are powerful technologies offering many advantages over bulk measurements, including high resolution, well controlled experimental conditions, low analyte volume requirement, short analysis time and low cost. ![]() This general principle of isoelectric focusing (IEF) can be applied using capillaries IEF, 1,16 gel slabs IEF 17 or macro scale Free Flow Isoelectric Focusing (FFIEF). One possible way to characterise such pH gradients is the use of a set of molecular markers, such as fluorescently labelled peptides or proteins, of known pI 1 to which the pI investigated sample can be added. 15 A significant challenge underlying this approach is the requirement to generate and maintain a spatially stable pH gradient of a known and well controlled magnitude. 14 Proteins that are then introduced into the system migrate in the gradient until they reach their pI and start to precipitate. 13 However, the predominant way to separate proteins and investigate their pI is isoelectric focusing (IEF), in which a pH gradient is generated across a chamber when an electric field is applied. 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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