Miniaturization of hydrolase assays in thermocyclers

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 th...

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Bibliographic Details
Published in:Analytical biochemistry 2013-03, Vol.434 (1), p.39-43
Main Authors: Lucena, Severino A., Moraes, Caroline S., Costa, Samara G., de Souza, Wanderley, Azambuja, Patrícia, Garcia, Eloi S., Genta, Fernando A.
Format: Article
Language:English
Subjects:
absorbance
Amylase
Amylases - metabolism
Assay
beta-glucanase
Bicinchoninic acid (BCA)
bioactive properties
Dinitrosalicylic acid (DNS)
enzymatic reactions
Enzyme
enzyme activity
Enzyme Assays - instrumentation
Enzyme Assays - methods
evaporation
Glucan 1,3-beta-Glucosidase - metabolism
glycosidases
Humans
Hydrolase
Kinetics
Miniaturization - instrumentation
polymerase chain reaction
Quinolines - chemistry
reducing sugars
Salicylates - chemistry
Saliva - enzymology
Starch - metabolism
temperature
β-1,3-Glucanase
β-Glycosidase
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Summary: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.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2012.10.032