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Abstract:

A high-throughput screening was developed for the detection of phosphatase activity in bacterial colonies. Unlike other methods, the current procedure can be applied to any phosphatase because it uses physiological substrates and detects the compelled product of all phosphatase reactions, that is, orthophosphate. In this method, substrates diffuse from a filter paper across a nitrocellulose membrane to bacterial colonies situated on the opposite face, and then reaction products flow back to the paper. Finally, a colorimetric reagent discloses the presence of orthophosphate in the filter paper. We validated the performance of this assay with several substrates and experimental conditions and with different phosphatases, including a library of randomly mutagenized rapeseed chloroplast fructose-1,6-bisphosphatase. This procedure could be extended to other enzymatic activities provided that an appropriate detection of reaction products is available. © 2004 Elsevier Inc. All rights reserved.

Registro:

Documento: Artículo
Título:A high-throughput screening for phosphatases using specific substrates
Autor:Senn, A.M.; Wolosiuk, R.A.
Filiación:Instituto Leloir, Fac. de Ciencias Exactas Y Naturales, Universidad de Buenos Aires, Patricias Argentinas 435, C1405BWE Buenos Aires, Argentina
Palabras clave:Enzyme activity; High-throughput screening; Phosphatases; Transformed bacteria; fructose bisphosphatase; phosphatase; article; bacterium colony; biofilter; controlled study; enzyme activity; enzyme mechanism; enzyme specificity; high throughput screening; mutagenesis; nonhuman; physiology; priority journal; rapeseed; Bacteria (microorganisms); Sinapis arvensis
Año:2005
Volumen:339
Número:1
Página de inicio:150
Página de fin:156
DOI: http://dx.doi.org/10.1016/j.ab.2004.12.021
Título revista:Analytical Biochemistry
Título revista abreviado:Anal. Biochem.
ISSN:00032697
CODEN:ANBCA
CAS:fructose bisphosphatase, 9001-52-9; phosphatase, 9013-05-2
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00032697_v339_n1_p150_Senn

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Citas:

---------- APA ----------
Senn, A.M. & Wolosiuk, R.A. (2005) . A high-throughput screening for phosphatases using specific substrates. Analytical Biochemistry, 339(1), 150-156.
http://dx.doi.org/10.1016/j.ab.2004.12.021
---------- CHICAGO ----------
Senn, A.M., Wolosiuk, R.A. "A high-throughput screening for phosphatases using specific substrates" . Analytical Biochemistry 339, no. 1 (2005) : 150-156.
http://dx.doi.org/10.1016/j.ab.2004.12.021
---------- MLA ----------
Senn, A.M., Wolosiuk, R.A. "A high-throughput screening for phosphatases using specific substrates" . Analytical Biochemistry, vol. 339, no. 1, 2005, pp. 150-156.
http://dx.doi.org/10.1016/j.ab.2004.12.021
---------- VANCOUVER ----------
Senn, A.M., Wolosiuk, R.A. A high-throughput screening for phosphatases using specific substrates. Anal. Biochem. 2005;339(1):150-156.
http://dx.doi.org/10.1016/j.ab.2004.12.021