DC FieldValueLanguage
dc.contributor.authorWurm, Matthias-
dc.contributor.authorIlhan, Sibel-
dc.contributor.authorJandt, Uwe-
dc.contributor.authorZeng, An-Ping-
dc.date.accessioned2019-03-13T14:30:10Z-
dc.date.available2019-03-13T14:30:10Z-
dc.date.issued2019-04-
dc.identifier.citationAnalytical biochemistry (570): 32-42 (2019-04)de_DE
dc.identifier.issn0003-2697de_DE
dc.identifier.urihttp://hdl.handle.net/11420/2157-
dc.description.abstractUtilizing flow cytometry to monitor progress of bulk biochemical reactions and concentration of chemical species normally relies on the utilization of cells carrying intrinsic fluorescence or modified beads. We present a method for a simple measurement of the fluorescent marker molecule fluorescein and GFPuv in bulk solutions with high sensitivity using a CytoFLEX flow cytometer and without the need for modified beads. Polystyrene beads were used to trigger measurements based on their high scatter signal, to detect the fluorescence signal from two different fluorophores present in the sample solution. We report sensitivities of 33 pg/mL for fluorescein and 50 ng/mL for GFPuv. This method is comparable in sensitivity to a typical spectrometric fluorescence assay tested with fluorescein, and approximately ten times more sensitive for the measurement of GFPuv. PEG was added to the sample at a low concentration of 0.001% (w/v) to block unspecific GFPuv binding to the beads. The method was further applied to measure the GFPuv concentration in crude cell lysate samples used for cell free protein expression. An advantage of this method over spectrometric assays is the ability to differentiate signal subpopulations in the sample based on their individual fluorescence intensities.en
dc.description.sponsorshipThe authors acknowledge partial funding by the German Federal Ministry of Education and Research (BMBF , grant 031B0222 ).de_DE
dc.language.isoende_DE
dc.relation.ispartofAnalytical biochemistryde_DE
dc.subjectDimerizationde_DE
dc.subjectFlow cytometryde_DE
dc.subjectFluorescence measurementde_DE
dc.subjectGreen fluorescent proteinde_DE
dc.titleDirect and highly sensitive measurement of fluorescent molecules in bulk solutions using flow cytometryde_DE
dc.typeArticlede_DE
dc.type.diniarticle-
dcterms.DCMITypeText-
tuhh.abstract.englishUtilizing flow cytometry to monitor progress of bulk biochemical reactions and concentration of chemical species normally relies on the utilization of cells carrying intrinsic fluorescence or modified beads. We present a method for a simple measurement of the fluorescent marker molecule fluorescein and GFPuv in bulk solutions with high sensitivity using a CytoFLEX flow cytometer and without the need for modified beads. Polystyrene beads were used to trigger measurements based on their high scatter signal, to detect the fluorescence signal from two different fluorophores present in the sample solution. We report sensitivities of 33 pg/mL for fluorescein and 50 ng/mL for GFPuv. This method is comparable in sensitivity to a typical spectrometric fluorescence assay tested with fluorescein, and approximately ten times more sensitive for the measurement of GFPuv. PEG was added to the sample at a low concentration of 0.001% (w/v) to block unspecific GFPuv binding to the beads. The method was further applied to measure the GFPuv concentration in crude cell lysate samples used for cell free protein expression. An advantage of this method over spectrometric assays is the ability to differentiate signal subpopulations in the sample based on their individual fluorescence intensities.de_DE
tuhh.publisher.doi10.1016/j.ab.2019.01.006-
tuhh.publication.instituteBioprozess- und Biosystemtechnik V-1de_DE
tuhh.type.opus(wissenschaftlicher) Artikel-
tuhh.institute.germanBioprozess- und Biosystemtechnik V-1de
tuhh.institute.englishBioprozess- und Biosystemtechnik V-1de_DE
tuhh.gvk.hasppnfalse-
dc.type.driverarticle-
dc.type.casraiJournal Article-
tuhh.container.volume570de_DE
tuhh.container.startpage32de_DE
tuhh.container.endpage42de_DE
dc.relation.projectBasistechnologien Nachwuchsgruppe: Multiskalige Modellierung und Modifikation von Multienzymkomplexen als Basistechnologie für zellfreie Reaktionskaskaden-
item.grantfulltextnone-
item.creatorGNDWurm, Matthias-
item.creatorGNDIlhan, Sibel-
item.creatorGNDJandt, Uwe-
item.creatorGNDZeng, An-Ping-
item.openairecristypehttp://purl.org/coar/resource_type/c_6501-
item.fulltextNo Fulltext-
item.openairetypeArticle-
item.creatorOrcidWurm, Matthias-
item.creatorOrcidIlhan, Sibel-
item.creatorOrcidJandt, Uwe-
item.creatorOrcidZeng, An-Ping-
item.languageiso639-1en-
item.cerifentitytypePublications-
crisitem.author.deptBioprozess- und Biosystemtechnik V-1-
crisitem.author.deptBioprozess- und Biosystemtechnik V-1-
crisitem.author.deptBioprozess- und Biosystemtechnik V-1-
crisitem.author.deptBioprozess- und Biosystemtechnik V-1-
crisitem.author.orcid0000-0001-8221-5176-
crisitem.author.orcid0000-0001-9768-7096-
crisitem.author.parentorgStudiendekanat Verfahrenstechnik-
crisitem.author.parentorgStudiendekanat Verfahrenstechnik-
crisitem.author.parentorgStudiendekanat Verfahrenstechnik-
crisitem.author.parentorgStudiendekanat Verfahrenstechnik-
crisitem.project.funderBundesministerium für Bildung und Forschung-
crisitem.project.funderid501100002347-
crisitem.project.funderrorid04pz7b180-
crisitem.project.grantno031A128-
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