In-gel precipitation of enzymatically released phosphate
2004
Тип документа:
Чланак у часопису (Објављена верзија)
,
© 2004 Elsevier Inc.
Метаподаци
Приказ свих података о документуАпстракт:
The phosphate precipitation reaction using ammonium molybdate and triethylamine under low pH has been applied to gel-based assays for detecting phosphate-releasing enzymes. The sensitivity of the assay is 10 pmol Pi/mm2 of 1.5-mm-thick gel. The assay is applicable to enzymes with a wide range of optimal pH, from acid (pH 4.5) to alkaline phosphatase (pH 9.7), and to enzymes that use acid-labile substrates such as apyrase and glutamine synthetase. Using a negative staining approach, maltose phosphorylase, a phosphate-consuming enzyme, can also be detected. The assay was used to detect glutamine synthetase isoforms, separated by nondenaturing polyacrylamide gel electrophoresis from crude maize extracts. For downstream applications such as staining gels for proteins, the gels with precipitate should be incubated in 10 mM dithiothreitol or β-mercaptoethanol until the precipitate is dissolved and then thoroughly washed in water. In comparison to calcium phosphate precipitation or the phosphomolybdate–malachite green method, this method is more sensitive. It is a very simple, rapid, versatile, reproducible, and inexpensive method that could be a useful tool in enzymological studies.
Кључне речи:
Acid phosphatase; Alkaline phosphatase; Ammonium molybdate; Apyrase; Gel-based assay; Glutamine synthetase; Inorganic phosphate; Maltose phosphorylase; Native PAGE; Orthophosphate; Precipitation; TriethylamineИзвор:
Analytical Biochemistry, 2004, 334, 2, 312-317Финансирање / пројекти:
- U.S. Department of Agriculture, agreement No. 2002-35100-12054
- ND-EPSCoR
DOI: 10.1016/j.ab.2004.07.010
ISSN: 0003-2697
PubMed: 15494139