Protocol of capillary isoelectric focusing to separate extremely acidic and basic proteins

Abstract

Protocol of capillary isoelectric focusing to separate extremely acidic and basic proteinsA new set-up was constructed for capillary isoelectric focusing (CIEF) involving a sampling capillary as a bypass fixed to the separation capillary. Sample solutions were subjected to a previously established pH gradient from the sample capillary. Besides performing conventional CIEF, the separation of ampholytic compounds with isoelectric points (pIs) beyond the pH gradient was carried out on this system. This method was termed as pH gradient driven electrophoresis (PGDE) and the basic mathematical expressions were derived to express the dynamic fundamentals. Proteins such as lysozyme, cytochrome C, and pepsin with pIs higher than 10 or below 3 were separated in a pH gradient provided by Pharmalyte (pH 3 -10). Finally, this protocol convincingly exhibited its potential in the separation of a solution of chicken egg white.

Recent advances in IEF in capillary tubes and microchips

“…Cunliffe et al 83 found an advantage of short separation time using CIEF under a pre‐steady‐state focusing process in detecting non‐covalent complex between a fluorescence‐labeled guanosine 5′‐triphosphate analogue and Ras‐like G protein. Yang et al 84 demonstrated pH‐gradient‐driven electrophoresis, in which very acidic or basic proteins were separated and detected when they migrated through a pH gradient that was preformed by the focused CAs. Montgomery et al 85 reported dynamic IEF in which the slope of the pH gradient formed in a capillary can be dynamically controlled by the voltage of the additional electrodes placed between the electrodes at the both ends.…”

“…For FS capillaries, a covalently attached linear polyacrylamide (LPA) coating developed by Hjertén 86 has been used continuously 83, 84, 87–93 and also has been applied to both PDMS chips 42 and glass/PDMS hybrid chips 47, 48. The coating procedure includes coupling of 3‐methacryloxypropyltrimethoxysilane to the silanol group on the wall and subsequent copolymerization with acrylamide.…”

“…The 30‐min injection enhanced sample loading by 10‐ to 100‐fold in comparison with the conventional sample loading, simply by filling the separation channel with the CA‐sample mixture. Yang et al 84 developed a new CIEF set‐up that uses a sampling capillary as a bypass fixed to the separation capillary. A sample solution was electrophoretically loaded from the same capillary onto a previously established pH gradient.…”

Capillary electrophoresis of proteins 2003–2005

“…Since CIEF only focuses proteins within a specified pH span, a method has been developed to also separate proteins with pI outside of the pH range of the used carrier ampholytes [145].…”

Bioanalytical Applications of Capillary Electrophoresis

“…CIEF has also been demonstrated using a sampling capillary as a bypass fixed to the separation capillary, providing an established pH gradient from the sample capillary for sample solutions (26). The separation of ampholytic compounds with isoelectric points beyond the pH gradient was demonstrated using this system and termed as pH gradient-driven electrophoresis.…”