Gradient-free determination of isoelectric points of proteins on chip

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Łapińska, U 
Saar, KL 
Yates, EV 
Herling, TW 
Müller, T 

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.

Animals, Cattle, Electrophoresis, Humans, Hydrogen-Ion Concentration, Isoelectric Point, Lab-On-A-Chip Devices, Lactoglobulins, Microfluidics, Myoglobin, Proteins, Serum Albumin, Bovine, Transferrin
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Physical Chemistry Chemical Physics
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Royal Society of Chemistry
Biotechnology and Biological Sciences Research Council (BB/J002119/1)
European Research Council (337969)
We acknowledge financial support from the Biotechnology and Biological Sciences Research Council, the Newman Foundation and the Elan Pharmaceuticals. The research leading to these results has also received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007- 2013) through the ERC grant PhysProt (agreement n1 337969).