A high-throughput screening for phosphatases using specific substrates
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, o...
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2005
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00032697_v339_n1_p150_Senn http://hdl.handle.net/20.500.12110/paper_00032697_v339_n1_p150_Senn |
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paper:paper_00032697_v339_n1_p150_Senn2023-06-08T14:24:03Z A high-throughput screening for phosphatases using specific substrates Senn, Alejandro Marcelo Wolosiuk, Ricardo Alejandro 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 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. Fil:Senn, A.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Wolosiuk, R.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 2005 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00032697_v339_n1_p150_Senn http://hdl.handle.net/20.500.12110/paper_00032697_v339_n1_p150_Senn |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
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 |
| spellingShingle |
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 Senn, Alejandro Marcelo Wolosiuk, Ricardo Alejandro A high-throughput screening for phosphatases using specific substrates |
| topic_facet |
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 |
| description |
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. |
| author |
Senn, Alejandro Marcelo Wolosiuk, Ricardo Alejandro |
| author_facet |
Senn, Alejandro Marcelo Wolosiuk, Ricardo Alejandro |
| author_sort |
Senn, Alejandro Marcelo |
| title |
A high-throughput screening for phosphatases using specific substrates |
| title_short |
A high-throughput screening for phosphatases using specific substrates |
| title_full |
A high-throughput screening for phosphatases using specific substrates |
| title_fullStr |
A high-throughput screening for phosphatases using specific substrates |
| title_full_unstemmed |
A high-throughput screening for phosphatases using specific substrates |
| title_sort |
high-throughput screening for phosphatases using specific substrates |
| publishDate |
2005 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00032697_v339_n1_p150_Senn http://hdl.handle.net/20.500.12110/paper_00032697_v339_n1_p150_Senn |
| work_keys_str_mv |
AT sennalejandromarcelo ahighthroughputscreeningforphosphatasesusingspecificsubstrates AT wolosiukricardoalejandro ahighthroughputscreeningforphosphatasesusingspecificsubstrates AT sennalejandromarcelo highthroughputscreeningforphosphatasesusingspecificsubstrates AT wolosiukricardoalejandro highthroughputscreeningforphosphatasesusingspecificsubstrates |
| _version_ |
1768546232591974400 |