Recycling isoelectric focusing: use of simple buffers

Bier, M.; Long, Terry D.

doi:10.1016/0021-9673(92)85530-7

Cited by 35 publications

(15 citation statements)

References 9 publications

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“…In comparison with batch mode-operating methods including recycling IEF [26][27][28], off-gel IEF and immobilized pI membrane methods [1,2,4,5], the suggested DF IEF offers continuous sampling and fraction collection. The large consumption of carrier electrolytes can be solved by design of dedicated carriers based on mixtures of simple defined buffers.…”

Section: Discussionmentioning

confidence: 97%

Divergent flow isoelectric focusing

Šlais

2008

Electrophoresis

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Continuous-flow isoelectric focusing (IEF) has the potential to be an important method in proteome analysis. The current devices do not fully use the advantages of IEF, because they do not utilize all its important features including changes in background conductivity during the focusing. A novel continuous-flow IEF method has been developed based on planar divergent flow and control of local electric field by conductivity of electrode electrolytes. A hydrophilized polypropylene nonwoven fabric was used for creation of flow and electric manifold, making the assembled device cheap, flexible and easy to set up and operate. By using the colored low-molecular-weight pI markers we demonstrated much higher speed of focusing in the new designed channel in comparison with a channel based on currently used rectangular geometry. The developed divergent-flow IEF combines the speed of micro flow channels with the separation efficiency and sample load capacity of preparative devices.

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Section: Discussionmentioning

confidence: 97%

Divergent flow isoelectric focusing

Šlais

2008

Electrophoresis

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“…These non-gel-based IEF methods can accommodate large sample volumes and amounts in contrast to gelbased IEF methods. Preparative IEF as a prefractionation technique was first proposed by Bier's laboratory [60,61]. To overcome problems associated with the original device, Righetti and colleagues [62] developed a multicompartment electrolyser in which each compartment was separated For analytical imaging separations, a portion of each first-dimension FFE-IEF fraction (50 µl/total volume ∼2 ml) was injected directly from the 96 deep-well plate using the Agilent 1100 HPLC equipped with a well-plate autosampler and samples collected automatically into a multi-plate fraction collection system by a polyacrylamide gel membrane, each with a defined pH.…”

Section: Prefractionation Approaches In Proteome Analysismentioning

confidence: 99%

Proteomic analysis of colorectal cancer: prefractionation strategies using two‐dimensional free‐flow electrophoresis

Moritz

Skandarajah

et al. 2005

Comp Funct Genom

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This review deals with the application of a new prefractionation tool, free-flow electrophoresis (FFE), for proteomic analysis of colorectal cancer (CRC). CRC is a leading cause of cancer death in the Western world. Early detection is the single most important factor influencing outcome of CRC patients. If identified while the disease is still localized, CRC is treatable. To improve outcomes for CRC patients there is a pressing need to identify biomarkers for early detection (diagnostic markers), prognosis (prognostic indicators), tumour responses (predictive markers) and disease recurrence (monitoring markers). Despite recent advances in the use of genomic analysis for risk assessment, in the area of biomarker identification genomic methods alone have yet to produce reliable candidate markers for CRC. For this reason, attention is being directed towards proteomics as a complementary analytical tool for biomarker identification. Here we describe a proteomics separation tool, which uses a combination of continuous FFE, a liquid-based isoelectric focusing technique, in the first dimension, followed by rapid reversed-phase HPLC (1–6 min/analysis) in the second dimension. We have optimized imaging software to present the FFE/RP-HPLC data in a virtual 2D gel-like format. The advantage of this liquid based fractionation system over traditional gel-based fractionation systems is the ability to fractionate large quantity protein samples. Unlike 2D gels, the method is applicable to both high-Mr proteins and small peptides, which are difficult to separate, and in the case of peptides, are not retained in standard 2D gels.

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“…They demonstrated that by filling the recycling electrophoretic apparatus with a concentrated solution of a single IEB of the appropriate pI value (or a binary mixture of two IEBs that had slightly different pI values and were poor CAs), a complex mixture of proteins could be separated into two fractions, the first containing the proteins with pI values lower than that of the IEB, the second containing the proteins with pI values higher than that of the IEB. They pointed out that "... (it) is projected that for optimal use of our method, it would be advantageous to have available ampholytes covering the pH scale at 0.1 pH intervals ..." [37]. In an alternative approach to preparative-scale electrophoretic separation of proteins, in an attempt to mitigate some of the limitations of conventional isoelectric trapping (IET) [39,40], pH-biased IET was introduced recently [41].…”

Section: Introductionmentioning

confidence: 99%

“…IEBs also received interest in the preparative-scale electrophoretic separation of proteins: Bier and Long used IEBs to generate "pH windows" [36] in a free-flow recycling IEF apparatus [37]. They demonstrated that by filling the recycling electrophoretic apparatus with a concentrated solution of a single IEB of the appropriate pI value (or a binary mixture of two IEBs that had slightly different pI values and were poor CAs), a complex mixture of proteins could be separated into two fractions, the first containing the proteins with pI values lower than that of the IEB, the second containing the proteins with pI values higher than that of the IEB.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and characterization of quaternary ammonium dicarboxylic acid isoelectric buffers and their use in pH-biased isoelectric trapping separations

2005

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Two approaches are described in this paper for the synthesis of isoelectric buffers that have pI values in the 1.5 < pI < 4.3 range. The first synthesis relies on the alkylation of existing aminodicarboxylic acids and recovery of the ampholyte as an inner salt. The second synthesis method forms low-pI ampholytes by reacting a secondary amine with two equivalents of an alkylester of a haloalkanecarboxylic acid, followed by hydrolysis of the intermediate in an alkaline solution and recovery of the ampholyte as an inner salt. The new ampholytes have been analytically characterized by capillary electrophoresis, high-resolution electrospray ionization-mass spectrometry, one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy, and X-ray crystallography. The isoionic solutions of the new ampholytes have high buffering capacity and conductivity, making them good pH biasers in the receiving stream in preparative-scale pH-biased isoelectric trapping separations.

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Recycling isoelectric focusing: use of simple buffers

Cited by 35 publications

References 9 publications

Divergent flow isoelectric focusing

Divergent flow isoelectric focusing

Proteomic analysis of colorectal cancer: prefractionation strategies using two‐dimensional free‐flow electrophoresis

Synthesis and characterization of quaternary ammonium dicarboxylic acid isoelectric buffers and their use in pH-biased isoelectric trapping separations

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