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Separation Protocol (separation + protocol)

Distribution by Scientific Domains

Life Sciences	50%
Chemistry	50%

Selected Abstracts

Improved Synthesis and Isolation of 2,- O -Methyladenosine: Effective and Scalable Enzymatic Separation of 2,/3,- O -Methyladenosine Regioisomers

EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 19 2009
Saúl Martínez-Montero
Abstract An efficient separation of a mixture of 2,/3,- O -methyladenosine regioisomers (1 + 2; 1:1) has been developed by selective enzymatic acylation using immobilized Pseudomonas cepacia lipase (PSL-C) in combination with acetonoxime levulinate as acyl donor. The 3,-hydroxy group of 2,- O -methyladenosine (1) was acylated with high selectivity (ca. 70,%), whereas an equal amount of 3,- O -methyladenosine (2) in the same solution resulted in minor acylation of 5,-hydroxy group (ca. 8,%). The differential behavior of both regioisomers towards enzymatic acylation allowed to develop a separation protocol. Upon extraction of the acylated products, the 3,- O -methyladenosine was isolated in 81,% yield and 97,% purity from the aqueous layer. Hydrolysis of acylated products in organic layer furnished 2,- O -methyladenosine in 67,% yield and 99,% purity. The separation process was successfully applied to the crude reaction mixture of methylated products (ca. 3:1 of 1/2) on 5-g scale. We also report on the use of methyl p -toluenesulfonate as a safe reagent for 2,- O -methylation of adenosine.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source]

A novel procedure for gentle isolation and separation of intact infected and uninfected protoplasts from the central tissue of Vicia faba L. root nodules

PLANT CELL & ENVIRONMENT, Issue 7 2003
E. PEITER
ABSTRACT The central tissue of Vicia faba L. root nodules is composed of cells infected with Rhizobium bacteroids and uninfected cells. For the study of various processes, such as plasma membrane transport, it is essential to separate both cell types. Initial attempts to isolate protoplasts according to protocols described in the literature resulted in non-spherical and osmotically inactive material, which is in agreement with previous descriptions. In the study reported herein, it was shown that the plasma membrane of non-spherical infected protoplasts is not intact. A new isolation and separation protocol was developed, based on dissection of the nodule prior to cell wall digestion, non-shaking digestion in hypertonic medium, and a combined procedure for release of protoplasts into slightly hypotonic medium and separation of protoplast fractions by isopycnic density gradient centrifugation. Infected and uninfected protoplasts that were isolated according to this protocol were spherical, osmotically active and excluded propidium iodide, confirming the intactness of their plasma membrane. The common fluorescein diacetate test was shown to be artefactual in infected cells, since viable bacteroids also stain in defective cells. Light and electron microscopic examination of infected protoplasts showed that protoplasts still contained starch after isolation and bacteroids in intact protoplasts had unusually high amounts of polyhydroxybutyrate. The vacuoles of infected protoplasts contained protein and membrane-enclosed structures, and were of non-acid pH; traits that are typical of protein storage vacuoles. [source]

Antibody-immobilized column for quick cell separation based on cell rolling

BIOTECHNOLOGY PROGRESS, Issue 2 2010
Atsushi Mahara
Abstract Cell separation using methodological standards that ensure high purity is a very important step in cell transplantation for regenerative medicine and for stem cell research. A separation protocol using magnetic beads has been widely used for cell separation to isolate negative and positive cells. However, not only the surface marker pattern, e.g., negative or positive, but also the density of a cell depends on its developmental stage and differentiation ability. Rapid and label-free separation procedures based on surface marker density are the focus of our interest. In this study, we have successfully developed an antiCD34 antibody-immobilized cell-rolling column, that can separate cells depending on the CD34 density of the cell surfaces. Various conditions for the cell-rolling column were optimized including graft copolymerization, and adjustment of the column tilt angle, and medium flow rate. Using CD34-positive and -negative cell lines, the cell separation potential of the column was established. We observed a difference in the rolling velocities between CD34-positive and CD34-negative cells on antibody-immobilized microfluidic device. Cell separation was achieved by tilting the surface 20 degrees and the increasing medium flow. Surface marker characteristics of the isolated cells in each fraction were analyzed using a cell-sorting system, and it was found that populations containing high density of CD34 were eluted in the delayed fractions. These results demonstrate that cells with a given surface marker density can be continuously separated using the cell rolling column. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source]

Quinaphos and Dihydro-Quinaphos Phosphine,Phosphoramidite Ligands for Asymmetric Hydrogenation

CHEMISTRY - A EUROPEAN JOURNAL, Issue 25 2010
Thomas Pullmann Dr.
Abstract New derivatives of the Quinaphos ligands and the related Dihydro-Quinaphos ligands based on the more flexible 1,2,3,4-tetrahydroquinoline backbone have been prepared and fully characterised. A general and straightforward separation protocol was devised, which allowed for the gram-scale isolation of the Ra,Sc and Sa,Rc diastereomers. These new phosphine,phosphoramidite ligands have been applied in the Rh-catalysed asymmetric hydrogenation of functionalised olefins with the achievement of excellent enantioselectivities (,99,%) in most cases and turnover frequency (TOF) values of up to ,20,000,h,1. These results substantiate the practical utility of readily accessible Quinaphos-type ligands, which belong to the most active and selective category of ligands for Rh-catalysed hydrogenation known to date. [source]