Design of a new, twelve-channel electrophoretic apparatus based on the Gradiflow technology

Ogle, David; Sheehan, Marian; Rumbel, Brendan; Gibson, Toby; Rylatt, Dennis B.

doi:10.1016/s0021-9673(02)01525-x

Cited by 11 publications

(11 citation statements)

References 11 publications

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“…Membrane‐based flow‐through cells have become the cores of several new types of preparative electrophoretic devices, such as membrane contactors 101, 102, ion‐exchange ultrafiltration membrane partitioned FF‐IEF systems 103 or membrane‐mediated Gradiflow electrophoresis systems 104, 105, all of them used mostly for size‐ or charge‐based separations of proteins. The most spread approach among these systems, Gradiflow technology, was applied to purification of monoclonal antibodies from ascitic fluid 106, 107, Fab fragments from monoclonal antibody papain digest 108, polyclonal antibodies from human and some animal sera 109 and basic protein avidin from chicken egg white 110.…”

Section: Advances and New Concepts In Ffementioning

confidence: 99%

From micro to macro: Conversion of capillary electrophoretic separations of biomolecules and bioparticles to preparative free‐flow electrophoresis scale

Kašička

2009

Electrophoresis

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This invited contribution in the special issue of Electrophoresis published in celebration of the 30th Anniversary of this journal reflects the impact of our milestone paper [Prusík, Z., Kasicka, V., Mudra, P., Stepánek, J., Smékal, O., Hlavácek, J., Electrophoresis 1990, 11, 932-936] in the area of conversion of microscale analytical and micropreparative CE separations of biomolecules and bioparticles into (macro)preparative free-flow electrophoresis (FFE) scale on the basis of a correlation between CE and FFE methods. In addition to the survey of advances in the relatively narrow field of CE-FFE correlation and CE-FFE conversion, a comprehensive review of the recent developments of micropreparative CE and (macro)preparative FFE techniques is also presented and applications of these techniques to micro- and (macro)preparative separations and purifications of biomolecules and bioparticles are demonstrated. The review covers the period since the year of publication of the above paper, i.e. ca. the last 20 years.

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Section: Advances and New Concepts In Ffementioning

confidence: 99%

From micro to macro: Conversion of capillary electrophoretic separations of biomolecules and bioparticles to preparative free‐flow electrophoresis scale

Kašička

2009

Electrophoresis

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“…As the approach is similar to electrophoresis, it is sometimes referred to as an electrophoretic membrane contactor, particularly if a larger porous membrane is used as the separation medium for protein separation [5][6][7][8]. One or a series of separation membranes with different pore sizes has been used to extract proteins from egg white [9] and whey [7], to extract peptides from protein hydrolysate [10,11,12], and to purify charged molecules with low molecular weight [13]. Bazinet and co-workers have studied the effect of the electric field [14][15][16], pH [17], ionic strength [4] and membrane physicochemical properties [18] on both the separation ability and membrane fouling within the EDFM process; but an understanding and optimization of the EDFM process is far from complete.…”

Section: Introductionmentioning

confidence: 99%

“…Gel membranes are commonly used as restriction membranes [9,20], where the current is carried by the ions in the electrolyte solution which swells the membrane structure. Neutral materials such as polyacrylamide (PAm) are typically selected for this purpose.…”

Section: Introductionmentioning

confidence: 99%

“…The cation exchange membranes (Neosepta CMX) and anion exchange membranes (Neosepta AMX) were purchased from Astom Corporation, Minato-ku, Tokyo, Japan. All the membranes were immersed in the buffer solution for more than 24 h before use.The choice of buffer is a major factor for ensuring the separation performance in the EDFM process and buffers of low conductivity are recommended[9,25]. A Tris-Hepes buffer prepared with 20mM of Tris-base, 10 mM of Hepes and 1 mM of Ethylenediaminetetraacetic acid disodium salt (EDTANa2) at pH of 8.00 was used in both the separation and the electrode compartments.…”

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The effect of restriction membranes on mass transfer in an electrodialysis with filtration membrane process

Deng

Chen

Gras

et al. 2017

Journal of Membrane Science

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In an electrodialysis with filtration membrane (EDFM) separation process, a pair of restriction membranes is used to prevent leakage of molecules into the electrode solution. Driven by the electrical potential gradient, ions transport across these membranes in different directions, resulting in concentration changes in the feed and permeate compartments respectively. However, the effects of these restriction membranes have not been systemically studied to date. In the present work, the performance of a non-charged polyacrylamide (PAm) membrane and two ion-exchange membranes were compared. PAm restriction membranes were found to give the most stable operation, with only slight concentration and pH changes in the feed and permeate compartments. The configurations with either cation or anion exchange membranes showed more significant changes in electrolyte concentration in opposing directions but the same pH trend, as the current density increased. The configuration with cation exchange membranes as restriction membranes resulted in the highest protein recovery, while the use of anion exchange membranes would appear as the best choice for separation of a protein with net positive charge. The results of this work provide important insights for the design and operation of EDFM processes and a better understanding of the transport phenomena that occur during the separation of bioactive compounds.

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“…43 In this apparatus, the chambers are separated by thin polyacrylamide gels containing ampholyte mixtures at specific pH values; this device is now commercially available (Zoom IEF Fractionator, Invitrogen). Another liquid-based IEF prefractionation technology, the Gradiflow system, 44 comprises recirculating hydraulic flow of the protein mixtures through two shallow separation compartments with an orthogonal electrophoretic transport of different proteins across a single separation membrane between the recirculating compartments. Unlike most other multicompartment electrokinetic analyzers, the Gradiflow technology allows the electrophoretic separation of proteins based upon both a protein's charge and molecular shape (size) properties.…”

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confidence: 99%

A Proteome Strategy for Fractionating Proteins and Peptides Using Continuous Free-Flow Electrophoresis Coupled Off-Line to Reversed-Phase High-Performance Liquid Chromatography

et al. 2004

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Extensive prefractionation is now considered to be a necessary prerequisite for the comprehensive analysis of complex proteomes where the dynamic range of protein abundances can vary from approximately 10(6) for cells to approximately 10(10) for tissues such as blood. Here, we describe a high-resolution 2D protein separation system that uses a continuous free-flow electrophoresis (FFE) device to fractionate complex protein mixtures by solution-phase isoelectric focusing (IEF) into 96 well-defined pools, each separated by approximately 0.02-0.10 pH unit depending on the gradient created, followed by rapid (approximately 6 min per analysis) reversed-phase high-performance liquid chromatography (RP-HPLC) of each FFE pool. Fractionated proteins are readily visualized in a virtual 2D format using software that plots protein loci, pI in the first dimension and relative hydrophobicity (i.e., RP-HPLC retention time) in the second dimension. By coupling a diode-array detector in line with a multiwavelength fluorescence detector, separated proteins can be monitored in the RP-HPLC eluent by both UV absorbance and intrinsic fluorescence simultaneously from a single experiment. Triplicate analyses of standard proteins using a pH 3-10 gradient conducted over a 3-day period revealed a high system reproducibility with a SD of 0.57 (0.05 pH unit) within the FFE pools and 0.003 (0.18 s) for protein retention times in the second-dimension RP-HPLC step. In addition, we demonstrate that the FFE-IEF/RP-HPLC separation strategy can also be applied to complex mixtures of low molecular weight compounds such as peptides. With the facile ability to measure the pH of the isoelectric focused pools, peptide pI values can be estimated and used to qualify peptide identifications made using either MS/MS sequencing approaches or pI discriminated peptide mass fingerprinting. The calculated peak capacity of this 2D liquid-based FFE-IEF/RP-HPLC system is 6720.

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Design of a new, twelve-channel electrophoretic apparatus based on the Gradiflow technology

Cited by 11 publications

References 11 publications

From micro to macro: Conversion of capillary electrophoretic separations of biomolecules and bioparticles to preparative free‐flow electrophoresis scale

From micro to macro: Conversion of capillary electrophoretic separations of biomolecules and bioparticles to preparative free‐flow electrophoresis scale

The effect of restriction membranes on mass transfer in an electrodialysis with filtration membrane process

A Proteome Strategy for Fractionating Proteins and Peptides Using Continuous Free-Flow Electrophoresis Coupled Off-Line to Reversed-Phase High-Performance Liquid Chromatography

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