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[Physical Chemistry Chemical Physics] Gradient-free determination of isoelectric points of proteins


Abstract:

The isoelectric point (pI) of a protein is a key characteristic that influences its overall electrostatic behaviour. 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. 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. 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. 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. 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.

Urszula Łapińska,a Kadi L. Saar,a Emma V. Yates,a Therese W. Herling,a Thomas Müller,ab Pavan K. Challa,a Christopher M. Dobsona and Tuomas P. J. Knowles*ac Author affiliations * Corresponding authors a Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK E-mail: tpjk2@cam.ac.uk Fax: +44 (0)1223 336362 Tel: +44 (0)1223 336344 b Fluidic Analytics, Unit 5 Chesterton Mill, French's Road, Cambridge CB4 3NP, UK c Department of Physics, Cavendish Laboratory, 19 J J Thomson Avenue, Cambridge CB3 0HE, UK

Link: http://pubs.rsc.org/en/content/articlelanding/2017/cp/c7cp01503h#!divAbstract

#08212017 #protein #electrostatic

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