Authors: Urszula Łapińska, Kadi L. Saar, Emma V. Yates, Therese W. Herling, Thomas Müller, Pavan K. Challa, Christopher M. Dobson, and Tuomas P. J. Knowles
Phys. Chem. Chem. Phys., 2017, 19, 23060-23067. DOI: 10.1039/C7CP01503H
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, Łapińska et al., 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, they 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.
Lapinska et al. using a microfluidic system built in house design a new technique to determine a protein's isoelectric point (pI) based on microfluidic free-flow electrophoresis (μFFE). The approach exploits temporal rather than spatial pH gradients. To demonstrate the effectiveness of this method the pI of 7 different proteins of known pI were tested; β-lactoglobulin, ribonuclease A, ovalbumin, human transferrin, ubiquitin and myoglobin.
The paper shows that this method is successful in determining the pI using this new technique without the requirement of generating and maintaining pH gradients which is often challenging for other techniques. The technique requires low voltages and low sample consumption. The paper also shows that using this technique it is possible to estimate the pI values for a wide range of proteins measuring at only two pH values, suggesting that this technique is rapid and accurate on small volume samples.
Figure 1: Shows a diagram of the microfluidic chip used to determine the pI of the target proteins without a spatial pH gradient and instead using a temporal gradient.
Therapeutic area: basic research