Automated flow-based anion-exchange method for high-throughput isolation and real-time monitoring of RuBisCO in plant extracts

Show simple item record Suárez Sánchez, Ruth Miró Lladó, Manuel Cerdà Martín, Víctor Perdomo López, Juan Alejandro Galmés Galmés, Jeroni 2018-09-21T12:08:47Z
dc.description.abstract [eng] In this work, a miniaturized, completely enclosed multisyringe-flow system is proposed for high-throughput purification of RuBisCO from Triticum aestivum extracts. The automated method capitalizes on the uptake of the target protein at 4ºC onto Q-Sepharose Fast Flow strong anion-exchanger packed in a cylindrical microcolumn (105 × 0.4 mm) followed by a stepwise ionic-strength gradient elution (0-0.8 mol/L NaCl) to eliminate concomitant extract components and retrieve highly purified RuBisCO. The manifold is furnished downstream with a flow-through diode-array UV/Vis spectrophotometer for real-time monitoring of the column effluent at the protein-specific wavelength of 280 nm to detect the elution of RuBisCO. Quantitation of RuBisCO and total soluble proteins in the eluate fractions were undertaken using polyacrylamide gel electrophoresis (PAGE) and the spectrophotometric Bradford assay, respectively. A comprehensive investigation of the effect of distinct concentration gradients on the isolation of RuBisCO and experimental conditions (namely, type of resin, column dimensions and mobile-phase flow rate) upon column capacity and analyte breakthrough was effected. The assembled set-up was aimed to critically ascertain the efficiency of preliminary batchwise pre-treatments of crude plant extracts (viz., polyethylenglycol (PEG) precipitation, ammonium sulphate precipitation and sucrose gradient centrifugation) in terms of RuBisCO purification and absolute recovery prior to automated anion-exchange column separation. Under the optimum physical and chemical conditions, the flow-through column system is able to admit crude plant extracts and gives rise to RuBisCO purification yields better than 75%, which might be increased up to 96 ± 9% with a prior PEG fractionation followed by sucrose gradient step.
dc.format application/pdf
dc.relation.isformatof Versió postprint del document publicat a:
dc.relation.ispartof Talanta, 2011, vol. 85, p. 1259-1266
dc.rights (c) Elsevier B.V., 2011
dc.subject.classification 54 - Química
dc.subject.classification 57 - Biologia
dc.subject.other 54 - Chemistry. Crystallography. Mineralogy
dc.subject.other 57 - Biological sciences in general
dc.title Automated flow-based anion-exchange method for high-throughput isolation and real-time monitoring of RuBisCO in plant extracts
dc.type info:eu-repo/semantics/article
dc.type info:eu-repo/semantics/acceptedVersion 2018-09-21T12:08:48Z info:eu-repo/date/embargoEnd/2075-01-01
dc.embargo 2075-01-01
dc.rights.accessRights info:eu-repo/semantics/embargoedAccess

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