Recycling isoelectric focusing and isotachophoresis

Abstract

Of all electrophoretic methods, isoelectric focusing offers the highest resolution and is best suited for preparative applications. Over the years, several instruments were developed for this purpose, all operating in free fluids, in the absence of gels or other supporting matrices. In such systems, the avoidance of gravity or electrically driven convections is essential. Successful stratagems for fluid control included rapid recycling or rotation, in combination with either fine porosity screens or narrow gaps between parallel plates. The most successful apparatus so far is the Rotofor, in which fluid is stabilized by combining horizontal rotation with fine porosity screen partitioning. Recycling isotachophoresis offers the potential of separating proteins at high concentration. A new concept of tangential electrophoresis is described. To optimize the use of these devices for protein separation, low molecular weight, biologically acceptable buffers of known composition are essential. The buffering system developed for this purpose comprises a series of binary buffers that cover the pH range in steps of 1 pH unit or less. The pH gradient can be custom-designed and is of remarkable stability in operation.

Prefractionation of Proteome by Liquid Isoelectric Focusing Prior to Two-Dimensional Liquid Chromatography Mass Spectrometric Identification

“…For example, the As a high-abundance protein, HS72_YEAST(CAI value 0.82) could be identified easily by capillary 2D-LC-ESI-MS/MS via introducing various derived peptides into the system. Although 9 SCX salt steps were used to fractionate the peptide mixtures from the total lysate of yeast, a number of peptides, even the same peptides from HS72_YEAST, were detected throughout of the 9 steps by MS. After the prefractionation of proteins from yeast, HS72_YEAST were clustered into limited fractions (e.g., fraction 25,30,29,28,27,26,24). Only in fraction 25 were peptides from HS72 detected in each of the 9 salt steps; in the other tubes, those peptides were only detected in limited salt steps (e.g., fraction/step/hits: 30/4/6, 29/2/2, 28/3/35, 27/8/ 55, 26/4/35, 24/8/119).…”

“…27 Up to now, several commercial instruments for solution IEF have been developed, including the Rotofor, RF3 (Recycling Free Flow Focusing protein fractionator), the Isoprime, and the IsoelectrIQ2. 9,[27][28][29][30][31][32][33][34][35][36][37][38][39][40]42 In this work, we have developed a method to separate proteins prior to protein digestion and 2D-LC-MS/ MS identification according to their pI, using LIEF in the RF3. Protein pre-fractionation diluted the high abundance proteins by distributing different forms of high abundance proteins into several fractions; the concentration of low abundance proteins could also be increased in the fractionated area, allowing easier detection.…”

Gel‐free IEF in a membrane‐sealed multicompartment cell for proteome prefractionation

“…Notwithstanding the advantages of proteome prefractionation in IPG-based separation processes (high precision in pH gradient engineering, very high resolution, retrieval of sample uncontaminated by carrier ampholytes (CAs)), separations in conventional IEF in soluble amphoteric buffers have also been adopted recently, especially in the Rotofor system (and in the mini-Rotofor version) [25]. The Rotofor is assembled from 20 sample chambers, separated by liquid-permeable nylon screens, except at the extremities, where cation-and anion-exchange membranes are placed against the anodic and cathodic compartments, respectively, so as to prevent diffusion within the sample chambers of noxious electrodic products.…”

Protein purification by Off-Gel electrophoresis

“…Some of them have been reviewed in the literature [10][11][12]. One of the most common preparative approaches to recycling free-flow electrophoresis is the Rotofor apparatus, commercialized by Bio-Rad (Hercules, CA, USA).…”

“…This device has been successfully applied to the preparative scale. A modification of this approach is the tangential electrophoretic apparatus from Bier [10]. Here, the different compartments are arranged in such a manner that an array of multi-channels is separated from a second array of multichannels slightly displaced through a single screen.…”