Protein adsorption to inner capillary walls creates a major obstacle in all applications of capillary electrophoresis involving protein samples. The problem is especially severe in kinetic capillary electrophoresis (KCE) techniques, which are used to study protein-ligand interactions at physiological conditions and, thus, cannot utilize extreme pH. A variety of coatings exist to reduce protein adsorption in CE, each expressing a unique surface chemistry that interacts with individual proteins differently. Here we introduce a simple pressure-based method for the qualitative assessment of protein adsorption that can facilitate the direct antiadhesive ranking of several coatings toward a protein of interest. In this approach, a short plug of the protein is injected into a capillary and propagated through with a pressure low enough to ensure adequate Taylor dispersion. The experiment is performed with a nonmodified commercial instrument in a pseudo-two-detector approach. The two detectors are mimicked by using two different distances from the capillary inlet to a single detector. If the peak area and shape do not change with changing distance, the protein does not adsorb appreciably, while a decreasing peak area with increasing distance infers inner surface adsorption. The magnitude change of the peak area between the two distances along with the overall peak shape is used to gauge the extent of protein adsorption. By using this method, we ranked antiadhesive properties of different wall chemistries for a series of proteins. The described method will be useful for optimizing protein analysis by CE and, in particular, for KCE experiments that investigate how proteins interact with their respective ligands.

Protein adsorption to the inner capillary wall hinders the use of kinetic capillary electrophoresis (KCE) when studying noncovalent protein-ligand interactions. Permanent and dynamic capillary coatings have been previously reported to alleviate much of the problems associated with protein adsorption. The characteristic limitations associated with permanent and dynamic coatings motivated us to look at a third type of coating - semipermanent. Here, we demonstrate that a semipermanent capillary coating, designed by Lucy and co-workers, comprised of dioctadecyldimethylammonium bromide (DODAB) and polyoxyethylene (POE) stearate, greatly reduces protein adsorption at physiological pH - a necessary requirement for KCE. The coating (i) does not inhibit protein-DNA complex formation, (ii) prevents the adsorption of the analytes, and (iii) supports an electoosmotic flow required for many applications of KCE. The coating was tested in three physiological buffers using a well-known DNA aptamer and four proteins that severely bind to bare silica capillaries as standards. For every protein, a condition was found under which the semipermanent coating effectively suppresses protein adhesion. While no coating can completely prevent the adsorption of all proteins, our findings suggest that the DODAB/POE stearate coating can have a broad impact on CE at large, as it prevents the absorption of several well studied, highly adhesive proteins at physiological pH.

Methods of kinetic capillary electrophoresis (KCE) facilitate highly efficient selection of DNA aptamers for protein targets. The inability to detect native proteins at low concentrations in capillary electrophoresis creates, however, a significant obstacle for many important protein targets. Here we suggest that protein labeling with new Chromeo dyes can help to overcome this obstacle. By labeling a number of proteins with Chromeo P503, we show that the labeling procedure enables accurate detection of proteins in CE without significantly affecting their electrophoretic mobility or their ability to bind DNA. Moreover, Chromeo P503 does not appear to label the amino-groups of buffer components to a significant extent, making the labeling procedure compatible with a large number of selection and run buffers. Fluorescent labeling of protein targets with Chromeo dyes empowers selection of aptamers by KCE methods and promises to increase the rate at which aptamers for new targets are being developed and introduced in various applications.