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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| Acceso en línea: | http://hdl.handle.net/20.500.12110/paper_00032697_v339_n1_p150_Senn |
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todo:paper_00032697_v339_n1_p150_Senn2023-10-03T13:55:51Z A high-throughput screening for phosphatases using specific substrates Senn, A.M. Wolosiuk, R.A. 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. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar 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, A.M. Wolosiuk, R.A. 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. |
| format |
JOUR |
| author |
Senn, A.M. Wolosiuk, R.A. |
| author_facet |
Senn, A.M. Wolosiuk, R.A. |
| author_sort |
Senn, A.M. |
| 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 |
| url |
http://hdl.handle.net/20.500.12110/paper_00032697_v339_n1_p150_Senn |
| work_keys_str_mv |
AT sennam ahighthroughputscreeningforphosphatasesusingspecificsubstrates AT wolosiukra ahighthroughputscreeningforphosphatasesusingspecificsubstrates AT sennam highthroughputscreeningforphosphatasesusingspecificsubstrates AT wolosiukra highthroughputscreeningforphosphatasesusingspecificsubstrates |
| _version_ |
1782024243542753280 |