Please use this identifier to cite or link to this item: https://www.arca.fiocruz.br/handle/icict/11863
Title: Miniaturization of hydrolase assays in thermocyclers
Authors: Lucena, Severino A.
Moraes, Caroline S.
Costa, Samara G.
Souza, Wanderley de
Azambuja, Patrícia
Garcia, Eloi S.
Genta, Fernando A.
Affilliation: Instituto Nacional de Metrologia, Qualidade e Tecnologia. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia. Departamento de Entomologia Molecular. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Instituto de Tecnologia Imunobiológica. Rio de Janeiro, RJ, Brasil.
Instituto Nacional de Metrologia, Qualidade e Tecnologia. Rio de Janeiro, RJ, Brasil
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia. Departamento de Entomologia Molecular. Rio de Janeiro, RJ, Brasil.
Instituto Nacional de Metrologia, Qualidade e Tecnologia. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia. Departamento de Entomologia Molecular. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia. Departamento de Entomologia Molecular. Rio de Janeiro, RJ, Brasil.
Abstract: We 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.
Keywords: Hydrolase
Enzyme
Assay
Amylase
B-1,3-Glucanase
β-Glycosidase
Dinitrosalicylic acid (DNS)
Bicinchoninic acid (BCA)
DeCS: Hidrolase
Enzima
Ensaio
Amilases
Glucosidase
Glucanase
Issue Date: 2013
Publisher: Elsevier
Citation: LUCENA, Severino A. et al. Miniaturization of hydrolase assays in thermocyclers. Analytical Biochemistry, v. 434, n. 1, p. 39-43, Mar. 2013.
DOI: 10.1016/j.ab.2012.10.032
ISSN: 0003-2697
Copyright: restricted access
Appears in Collections:Biomanguinhos - Artigos de Periódicos
IOC - Artigos de Periódicos

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