Download PDF by Senta Reichelt (eds.): Affinity Chromatography: Methods and Protocols

By Senta Reichelt (eds.)

ISBN-10: 149392446X

ISBN-13: 9781493924462

ISBN-10: 1493924478

ISBN-13: 9781493924479

The goal of this version is to introduce the newbie to the fundamentals of affinity chromatography and supply sensible wisdom for the advance of affinity separation protocols. Affinity Chromatography: tools and Protocols, 3rd Edition courses readers via new cutting-edge protocols, molecular modelling, and the examine of ligand-target interactions. Written within the winning Methods in Molecular Biology sequence structure, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, with ease reproducible protocols, and notes on troubleshooting and heading off recognized pitfalls.

Authoritative and simply accessible, Affinity Chromatography: tools and Protocols, 3rd Edition is designed as an invaluable source for these attracted to the speedy and quantitative isolation of biomolecules with excessive purity.

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The solution of the dye in N,N-dimethylformamide must be prepared carefully, assuring the complete dissolution of the dye. However, this is not always easy to check because of the very dark blue color of the solution. An option is to try to detect the presence of suspended shiny particles of the dye. Care must be taken to ensure that no precipitation of the dye occurs when this solution is added to the Sepharose suspension. 12. Perform the orbital agitation at 240 rpm. During this period, the mixture should be covered with aluminum foil to prevent exposure to light and avoid photodegradation of the dye.

During this period, the mixture should be covered with aluminum foil to prevent exposure to light and avoid photodegradation of the dye. 13. Again this washing step should be performed under controlled reduced pressure, so that the gel can be gently swirled with a spatula to assure an efficient washing without damage to the gel structure. Each organic solvent must be used until no more unbounded dye is removed. Methanol is the solvent that removes the dye more efficiently. Water is necessary to transfer the gel from the organic to an inorganic phase.

1007/978-1-4939-2447-9_3, © Springer Science+Business Media New York 2015 35 36 Cunxi Wang et al. reported that Reactive brown 10, along with other reactive dyes, can be used for purification of glycosyltransferases [3]. In addition, reactive dyes are stable, easy to be immobilized, inexpensive, and commercially available. Phosphinothricin N-acetyltransferase (PAT) is an enzyme that acetylates the free NH2 group of L-phosphinothricin (L-PPT) in the presence of acetyl-CoA as a co-substrate [9, 10].

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Affinity Chromatography: Methods and Protocols by Senta Reichelt (eds.)

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