Automated free‐solution isotachophoresis: Instrumentation and fractionation of human serum proteins

Abstract

An automated free-solution isotachophoresis system (FS-ITP) for preparative fractionation of biopolymers is described, operated in a batch mode. The dimension of the separation chamber allows an up to 1200-fold higher sample load compared to separation in capillaries of 180 microm inner diameter as used in analytical capillary isotachophoresis (C-ITP). The preparative capacity of the system is within the milligram range. The method is fully compatible with analytical C-ITP, which is essential for preparative-scale isotachophoresis with regard to optimization of electrolyte systems and the search for suitable spacers. As a model application the fractionation of human serum proteins is reported. The collected fractions were analyzed by C-ITP and agarose gel electrophoresis.

Relation between insulin resistance and fast-migrating LDL subfraction as characterized by capillary isotachophoresis

“…Capillary isotachophoresis (cITP) is a new technique for separating plasma lipoprotein subfractions based on their electric charges (14)(15)(16)(17)(18). The two cITP LDL subfractions, fast-migrating (f) and slow-migrating (s) LDL, represent the LDL( Ϫ ) and major LDL subfractions, respectively.…”

Preparation of Golgi subfractions with free‐solution isotachophoresis: Analysis of sphingomyelin synthesis in Golgi subfractions from rat liver

“…By introducing preparative FS-ITP to generate subcellular membrane fractions, microscale fractions were able to be sucessfully prepared with a device specifically designed for the retrieval of all types of cellular membranes or soluble biological material [3 11. The preparative ITP system used here is based on a support-free "thin-film" electrophoresis device that works in batch mode and uses buffer solutions with increased viscosities [32]. For this reason, biologically inert HPMC was added to the buffers, thus lending additional stability to sample zones.…”

“…The design of the FS-ITP device and running conditions are described in detail in a separate manuscript [32]. Briefly, a total of 300 pL of an unstacked Golgi fraction supplemented with appropriate additives (Table 1) was cautiously injected into the interface between leading and terminating electrolytes in the separation chamber.…”

“…However, FFE leads to an excessive dilution of the sample components during their separation process due to the large quantities of electrophoresis buffer used. In an attempt to circumvent the difficulties associated with FFE, a new batch technique, preparative free-solution isotachophoresis (FS-ITP), was developed [32]. It improves the resolution pattern and reduces the dilution of the collected fractions.…”

Exceeding 20 000‐fold concentration of protein by the on‐line isotachophoresis concentration in poly(methyl methacrylate) microchip

“…In MCE when the voltage was switched from injection to separation, the t d appeared because of the low-mobility of TE and we could get different t d values when we used different TEs: the lower the electrophoretic mobility of TE, the longer the t d of sample, and the larger the concentration effect. These six TEs we used have different electrophoretic mobility(u), with the following order [30].…”