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Automated Solid-Phase Protein Modification with Integrated Enzymatic Digest for Reaction Validation: Application of a Compartmented Microfluidic Reactor for Rapid Optimization and Analysis of Protein Biotinylation

Fraas, Regina 1; Diehm, Juliane 1; Franzreb, Matthias ORCID iD icon 1
1 Institut für Funktionelle Grenzflächen (IFG), Karlsruher Institut für Technologie (KIT)


Protein modification by covalent coupling of small ligands or markers is an important prerequisite for the use of proteins in many applications. Well-known examples are the use of proteins with fluorescent markers in many in vivo experiments or the binding of biotinylated antibodies via biotin–streptavidin coupling in the frame of numerous bioassays. Multiple protocols were established for the coupling of the respective molecules, e.g., via the C and N-terminus, or via cysteines and lysines exposed at the protein surface. Still, in most cases the conditions of these standard protocols are only an initial guess. Optimization of the coupling parameters like reagent concentrations, pH, or temperature may strongly increase coupling yield and the biological activity of the modified protein. In order to facilitate the process of optimizing coupling conditions, a method was developed which uses a compartmented microfluidic reactor for the rapid screening of different coupling conditions. In addition, the system allows for the integration of an enzymatic digest of the modified protein directly after modification. In combination with a subsequent MALDI-TOF analysis of the resulting fragments, this gives a fast and detailed picture not only of the number and extent of the generated modifications but also of their position within the protein sequence. ... mehr

Verlagsausgabe §
DOI: 10.5445/IR/1000081425
Veröffentlicht am 27.03.2018
DOI: 10.3389/fbioe.2017.00072
Zitationen: 1
Zitationen: 2
Cover der Publikation
Zugehörige Institution(en) am KIT Institut für Funktionelle Grenzflächen (IFG)
Publikationstyp Zeitschriftenaufsatz
Publikationsjahr 2017
Sprache Englisch
Identifikator ISSN: 2296-4185
KITopen-ID: 1000081425
HGF-Programm 47.02.06 (POF III, LK 01) Zellpopul.auf Biofunk.Oberflächen IFG
Erschienen in Frontiers in Bioengineering and Biotechnology
Verlag Frontiers Media SA
Band 5
Seiten Article: 72
Bemerkung zur Veröffentlichung Gefördert durch den KIT-Publikationsfonds
Vorab online veröffentlicht am 13.11.2017
Nachgewiesen in Scopus
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