| Author | Lucena, Severino A. | |
| Author | Moraes, Caroline S. | |
| Author | Costa, Samara G. | |
| Author | Souza, Wanderley de | |
| Author | Azambuja, Patrícia | |
| Author | Garcia, Eloi S. | |
| Author | Genta, Fernando A. | |
| Access date | 2015-10-02T12:45:03Z | |
| Available date | 2015-10-02T12:45:03Z | |
| Document date | 2013 | |
| Citation | LUCENA, Severino A. et al. Miniaturization of hydrolase assays in thermocyclers. Analytical Biochemistry, v. 434, n. 1, p. 39-43, Mar. 2013. | pt_BR |
| ISSN | 0003-2697 | pt_BR |
| URI | https://www.arca.fiocruz.br/handle/icict/11863 | |
| Language | eng | pt_BR |
| Publisher | Elsevier | pt_BR |
| Rights | restricted access | |
| Title | Miniaturization of hydrolase assays in thermocyclers | pt_BR |
| Type | Article | |
| DOI | 10.1016/j.ab.2012.10.032 | pt_BR |
| 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. | pt_BR |
| Affilliation | Instituto Nacional de Metrologia, Qualidade e Tecnologia. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. | pt_BR |
| Affilliation | 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. | pt_BR |
| Affilliation | Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Instituto de Tecnologia Imunobiológica. Rio de Janeiro, RJ, Brasil. | pt_BR |
| Affilliation | Instituto Nacional de Metrologia, Qualidade e Tecnologia. Rio de Janeiro, RJ, Brasil | pt_BR |
| Affilliation | 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. | pt_BR |
| Affilliation | 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. | pt_BR |
| Affilliation | 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. | pt_BR |
| Subject | Hydrolase | pt_BR |
| Subject | Enzyme | pt_BR |
| Subject | Assay | pt_BR |
| Subject | Amylase | pt_BR |
| Subject | B-1,3-Glucanase | pt_BR |
| Subject | β-Glycosidase | pt_BR |
| Subject | Dinitrosalicylic acid (DNS) | pt_BR |
| Subject | Bicinchoninic acid (BCA) | pt_BR |
| DeCS | Hidrolase | pt_BR |
| DeCS | Enzima | pt_BR |
| DeCS | Ensaio | pt_BR |
| DeCS | Amilases | pt_BR |
| DeCS | Glucosidase | pt_BR |
| DeCS | Glucanase | pt_BR |
