Separation of DNA restriction fragments by ion-exchange chromatography on FPLC columns Mono P and Mono Q

Westman, Elisabeth; Eriksson, Stig; Låås, Torgny; Pernemalm, Per-Åke; Sköld, Sven-Erik

doi:10.1016/0003-2697(87)90558-6

Cited by 46 publications

(28 citation statements)

References 19 publications

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“…15. For preparation of the EcoRI to NdeI pDN-AdML fragment used in promoter-specific transcription assays, pDN-AdML was digested with EcoRI and NdeI, and the reaction mixture was adjusted to 0.6 M NaCl and subjected to chromatography on 1 ml HiTrap Q HP (Amersham Pharmacia Biosciences) anion exchange column as described by Westman et al (18). Fractions containing the EcoRI to NdeI fragment of pDN-AdML were pooled, ethanol precipitated, and resuspended in 10 mM Tris⅐HCl͞1 mM EDTA, pH 8.0.…”

Section: Methodsmentioning

confidence: 99%

ELL-associated factors 1 and 2 are positive regulators of RNA polymerase II elongation factor ELL

Kong

Banks

Shilatifard

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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In human cells, the ELL family of transcription factors includes at least three members, which are all capable of stimulating the overall rate of elongation by RNA polymerase II by suppressing transient pausing by the enzyme at many sites along DNA. In this report, we identify the ELL-associated factors (EAF)1 and EAF2 as strong positive regulators of ELL elongation activity. Our findings provide insights into the structure and function of ELL family transcription factors, and they bring to light direct roles for the EAF proteins in regulation of RNA polymerase II transcription.elongation ͉ mRNA synthesis ͉ transcription ͉ transcription factor T he gene encoding the RNA polymerase II (pol II) elongation factor eleven-nineteen lysine-rich in leukemia (ELL) was first characterized as a fusion partner of the mixed lineage leukemia (MLL) gene in the (11, 19)(q23;p13.1) translocation in acute myeloid leukemia (1). The ELL protein has been shown to be capable of interacting with pol II and increasing its rate of transcript elongation in vitro by suppressing transient pausing by the enzyme (2, 3). Consistent with a role for ELL in controlling transcript elongation in vivo, Drosophila ELL colocalizes with pol II at transcriptionally active sites on polytene chromosomes, and evidence suggests that mutations in the gene encoding Drosophila ELL may preferentially affect synthesis of long transcripts (4).In addition to ELL, human cells have two ELL paralogs, ELL2 and ELL3, which can also stimulate the rate of elongation by pol II in vitro (5, 6). ELL and ELL2 are expressed in a wide variety of tissues in humans. ELL3, on the other hand, appears to be expressed predominantly in the testis (5, 6).A variety of ELL-interacting proteins have been identified through two-hybrid screens and biochemical purifications. Two of these proteins, EAF1 and EAF2 (7,8), have been shown to colocalize with ELL in the nucleus in a stippled pattern that resembles that of Cajal bodies, structures that are enriched in factors involved in transcription and mRNA processing (9). Interestingly, Cajal bodies are disrupted in cells carrying the MLL-ELL translocation (10).Suggesting that interactions between ELL and EAF1 may contribute to the leukemic phenotype of cells expressing the MLL-ELL chimera, the C-terminal domains of EAF1 and EAF2 share similarity with AF4 and ENL, other MLL translocation partners (7,8). In addition, expression of artificial MLL-EAF1 and MLL-EAF2 chimeras can immortalize hematopoetic progenitor cells and induce the development of acute myeloid leukemia in mice (7,8). Finally, and consistent with the possibility that EAF1 and EAF2 could play a role in transcriptional regulation, their C-terminal regions function as transcriptional activation domains when fused to the GAL4 DNA binding domain (7,8).In this article, we identify the EAF1 and EAF2 proteins as positive regulators of ELL elongation activity. Our findings provide insights into the mechanism of action of ELL family transcription factors, and they bring to light direct ro...

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Section: Methodsmentioning

confidence: 99%

ELL-associated factors 1 and 2 are positive regulators of RNA polymerase II elongation factor ELL

Kong

Banks

Shilatifard

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…The AEC surface may act as a compacting agent [16]. Finally differences in retention behaviour of AT and GC rich sequences indicate distinctions in binding characteristics according to the DNA sequence [17,18].…”

Section: Introductionmentioning

confidence: 99%

Adsorption of plasmid DNA on anion exchange chromatography media

Tarmann

Jungbauer²

2008

J of Separation Science

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Original PaperAdsorption of plasmid DNA on anion exchange chromatography media Anion exchange chromatography (AEC) is a useful and effective tool for DNA purification, but due to average pore sizes between 40 and 100 nm most AEC resins lack truly useful binding capacities for plasmid DNA (pDNA). Equilibrium binding capacities and uptake kinetics of AEC media including conventional media (Source 30 Q, Q Sepharose HP), a polymer grafted medium (Fractogel EMD DEAE (M)), media with large pores (Celbeads DEAE, PL SAX 4000 30 lm) and a monolithic medium (CIM-DEAE) were investigated by batch uptake or shallow bed experiments at two salt concentrations. Theoretical and experimental binding capacities suggest that the shape of the pDNA molecule can be described by a rod with a length to diameter ratio of 20:1 and that the molecule binds in upright position. The arrangement of DNA like a brush at the surface can be considered as entropy driven, kind of selfassembly process which is inherent to highly and uniformly charged DNA molecules. The initial phase of adsorption is very fast and levels off, associated with a change in mass transfer mechanism. Feed concentrations higher than 0.1 mg/mL pDNA pronounce this effect. Monolithic media showed the fastest adsorption rate and highest binding capacity with 13 mg pDNA per mL.

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“…Most of the stationary phases are based on microparticles in the 3-5 mm range. Although such phases have been successfully utilized as separation media for HPLC for more than three decades [6,7], the relatively large void volume between the packed particles seriously limits the separation efficiency of conventional granular packing materials, especially on coupling to methods permitting structural analysis such as mass spectrometry (MS). Mass transfer is considerably enhanced by using monolithic separation media [8][9][10], consisting of a single piece of a rigid polymer which has no interstitial volume but instead only an internal porosity consisting of micro-, meso-, and macropores [11][12][13][14][15][16].…”

Section: Stationary Phasesmentioning

confidence: 99%

Recent progress in high‐performance capillary bioseparations

et al. 2003

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Bioanalysis is a fast growing domain with new technological developments in the field of mass spectrometry, separation science, and bioinformatics. The central role of separation prior to detection and evaluation of analytes is indisputable. In fact, since the decipherment of the human genome via multichannel capillary electrophoresis in combination with laser-induced fluorescence [1], the analysis of the proteome, i.e., the type and amount of all proteins expressed at a certain time by the genome in a cell or tissue [2] became of central interest. Therefore, new fast and sensitive capillary liquid chromatographic, electrophoretic, and electrochromatographic separation techniques, suitable for high-throughput analysis of proteins, peptides and nucleic acids, are required.

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Separation of DNA restriction fragments by ion-exchange chromatography on FPLC columns Mono P and Mono Q

Cited by 46 publications

References 19 publications

ELL-associated factors 1 and 2 are positive regulators of RNA polymerase II elongation factor ELL

ELL-associated factors 1 and 2 are positive regulators of RNA polymerase II elongation factor ELL

Adsorption of plasmid DNA on anion exchange chromatography media

Recent progress in high‐performance capillary bioseparations

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