Electrofractionation: A technique for detecting and recovering biomolecules

Shain, Daniel H.; Yoo, Jakyoung; Slaughter, R.G.; Hayes, Scott; Ji, Tae H.

doi:10.1016/0003-2697(92)90275-c

Cited by 21 publications

(8 citation statements)

References 7 publications

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“…These systems used nonporous media for the separations. A technique termed electrofraction has been used for recovering DNA fragments and oligonucleotides after gel electrophoresis (Shain et al, 1992). Electrofraction is a form of gel electrophoresis coupled to a chromatography system for recovery of components eluting from the gel.…”

Section: Discussionmentioning

confidence: 99%

Preparative Concentration and Size Fractionation of DNA by Porous Media Using a Combination of Flow and Low Electric Field Strength

Cole¹

1997

Biotechnol. Prog.

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The retention of DNA by porous media during chromatography with an applied axial electric field was investigated. DNA was retained by negative electric fields in columns packed with Sephadex G-75 and G-25. A negative field was defined as the electric field orientation in which the direction of electrophoresis was opposing the direction of buffer flow (positive electrode at the column inlet). A positive field was not effective at retaining the DNA. The electric field strength required to retain the DNA was dependent upon the buffer flow rate. The retention of DNA using Sephadex G-25, a gel filtration medium with a higher degree of cross-linking, required higher electric fields than the more porous Sephadex G-75. A dilute DNA solution was concentrated at the inlet of the chromatography bed by an electric field. Mixtures of DNA restriction fragments were used to determine size-dependent retention. DNA was size-fractionated by varying the electric field strength and flow rate. At a given electric field strength and flow rate, the lower molecular weight DNA fragments were not as strongly retained as the higher molecular weight fragments. Decreasing the flow rate or increasing the electric field strength resulted in increased retention of the lower molecular weight DNA fragments. In this manner, by selecting a specific set of conditions (packing material, flow rate, and electric field strength), the molecular weight of DNA fragments retained by the column can be adjusted. Efficient separation of high molecular weight DNA from bovine serum albumin, a protein with high electrophoretic mobility, was demonstrated using a field of 2 V/cm in a column packed with Sephadex G-75.

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Section: Discussionmentioning

confidence: 99%

Preparative Concentration and Size Fractionation of DNA by Porous Media Using a Combination of Flow and Low Electric Field Strength

Cole¹

1997

Biotechnol. Prog.

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“…Mouse genomic DNA (100-200 jig) was digested with EcoRI and electrophoresed on a 1% low melt agarose gel (FMC genetic technology grade) at 1 V/cm to achieve adequate separation. The region containing 4 kb fragments was excised and the DNA was electroeluted as described previously (39). Southern analysis was performed on 1/10 of the sample to confirm that the correct DNA fragment was present.…”

Section: Sub-genomic Library Constructionmentioning

confidence: 99%

A novel initiator regulates expression of the nontissue-specific helix-loop-helix gene ME1

Shain¹,

Neuman²,

Zuber³

1995

Nucleic Acids Research

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The mouse ME1 gene (HEB, REB and GE1, homologues in human, rat and chick, respectively) is a member of the nontissue-specific helix-loop-helix (HLH) gene family that includes E2A, E2-2 and Drosophila daughterless. We have examined the factors that control ME1 gene expression. ME1 is a single copy gene that spans > or = 150 kb of DNA and contains > 10 exons. Transcription was directed by an unusual initiator element that contained a 13 bp poly d(A) tract flanked by palindromic and inverted repeat sequences. Both RNase protection and primer extension analyses mapped the ME1 transcriptional start site to the center of the 13 bp poly d(A) tract. The ME1 initiator and its proximal sequences were required for promoter activity, supported basal levels of transcription, and contributed to cell type-specific gene expression. Other cis-elements utilized by the TATA-less ME1 promoter included a cluster of Sp1 response elements, E-boxes and a strong repressor. Collectively, our results suggest that the ME1 initiator and other cis-elements in the proximal promoter play an important role in regulating ME1 gene expression.

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“…In the beginning till middle of the 1960s, different groups began already to develop the respective instruments [132,[167][168][169]. During the years, this system was improved stepwise, not only for SDS-PAGE but also for native complexes [170][171][172]. Further examples are listed in Table 1.…”

Section: Continuous Elution Devicesmentioning

confidence: 99%

Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels

Seelert

Krause

2008

Electrophoresis

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Due to its unmatched resolution, gel electrophoresis is an indispensable tool for the analysis of diverse biomolecules. By adaptation of the electrophoretic conditions, even fragile protein complexes as parts of intracellular networks migrate through the gel matrix under sustainment of their integrity. If the thickness of such native gels is significantly increased compared to the analytical version, also high sample loads can be processed. However, the cage-like network obstructs an in-depth analysis for deciphering structure and function of protein complexes and other species. Consequently, the biomolecules have to be removed from the gel matrix into solution. Several approaches summarized in this review tackle this problem. While passive elution relies on diffusion processes, electroelution employs an electric field to force biomolecules out of the gel. An alternative procedure requires a special electrophoresis setup, the continuous elution device. In this apparatus, molecules migrate in the electric field until they leave the gel and were collected in a buffer stream. Successful isolation of diverse protein complexes like photosystems, ATP-dependent enzymes or active respiratory supercomplexes and some other bioparticles demonstrates the versatility of preparative electrophoresis. After liberating particles out of the gel cage, numerous applications are feasible. They include elucidation of the individual components up to high resolution structures of protein complexes. Therefore, preparative electrophoresis can complement standard purification methods and is in some cases superior to them.

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Electrofractionation: A technique for detecting and recovering biomolecules

Cited by 21 publications

References 7 publications

Preparative Concentration and Size Fractionation of DNA by Porous Media Using a Combination of Flow and Low Electric Field Strength

Preparative Concentration and Size Fractionation of DNA by Porous Media Using a Combination of Flow and Low Electric Field Strength

A novel initiator regulates expression of the nontissue-specific helix-loop-helix gene ME1

Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels

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