Miniaturization of hydrolase assays in thermocyclers

dc.creatorLucena, Severino A.
dc.creatorMoraes, Caroline S.
dc.creatorCosta, Samara G.
dc.creatorSouza, Wanderley de
dc.creatorAzambuja, Patrícia
dc.creatorGarcia, Eloi S.
dc.creatorGenta, Fernando A.
dc.date2015-10-02T12:45:03Z
dc.date2015-10-02T12:45:03Z
dc.date2013
dc.date.accessioned2023-09-26T23:32:47Z
dc.date.available2023-09-26T23:32:47Z
dc.identifierLUCENA, Severino A. et al. Miniaturization of hydrolase assays in thermocyclers. Analytical Biochemistry, v. 434, n. 1, p. 39-43, Mar. 2013.
dc.identifier0003-2697
dc.identifierhttps://www.arca.fiocruz.br/handle/icict/11863
dc.identifier10.1016/j.ab.2012.10.032
dc.identifier.urihttps://repositorioslatinoamericanos.uchile.cl/handle/2250/8891733
dc.descriptionWe adapted the protocols of reducing sugar measurements with dinitrosalicylic acid and bicinchoninic acid for thermocyclers and their use in enzymatic assays for hydrolases such as amylase and β-1,3-glucanase. The use of thermocyclers for these enzymatic assays resulted in a 10 times reduction in the amount of reagent and volume of the sample needed when compared with conventional microplate protocols. We standardized absorbance readings from the polymerase chain reaction plates, which allowed us to make direct readings of the techniques above, and a β-glycosidase assay was also established under the same conditions. Standardization of the enzymatic reaction in thermocyclers resulted in less time-consuming temperature calibrations and without loss of volume through leakage or evaporation from the microplate. Kinetic parameters were successfully obtained, and the use of the thermocycler allowed the measurement of enzymatic activities in biological samples from the field with a limited amount of protein.
dc.formatapplication/pdf
dc.languageeng
dc.publisherElsevier
dc.rightsrestricted access
dc.subjectHydrolase
dc.subjectEnzyme
dc.subjectAssay
dc.subjectAmylase
dc.subjectB-1,3-Glucanase
dc.subjectβ-Glycosidase
dc.subjectDinitrosalicylic acid (DNS)
dc.subjectBicinchoninic acid (BCA)
dc.subjectHidrolase
dc.subjectEnzima
dc.subjectEnsaio
dc.subjectAmilases
dc.subjectGlucosidase
dc.subjectGlucanase
dc.titleMiniaturization of hydrolase assays in thermocyclers
dc.typeArticle


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