PMID: 8601320Apr 1, 1996Paper

Fluorometric and time-resolved immunofluorometric assays for protein-tyrosine phosphatase activity

Clinical Biochemistry
B Galvan, T K Christopoulos

Abstract

To develop sensitive nonisotopic assays for protein-tyrosine phosphatase (PTP) activity. The fluorometric assay is based on the fact that phosphotyrosine but not tyrosine forms highly fluorescent complexes with Tb3+. Thus, PTP activity can be followed by measuring the decrease of fluorescence due to hydrolysis of phosphotyrosine. The time-resolved immunofluorometric assay employs tyrosine-phosphorylated substrates, immobilized on microtitre wells. After incubation with PTP, the remaining phosphotyrosine residues are reacted with an antiphosphotyrosine antibody. The immunocomplexes formed are detected with an alkaline phosphatase (ALP)-labeled second antibody. The phosphate ester of 5' fluorosalicylate (FSAP) is used as substrate. The fluorosalicylate produced forms highly fluorescent complexes with Tb3+ - EDTA in alkaline solution. The fluorescence is measured with a time-resolved fluorometer. The truncated form of the T-cell protein tyrosine phosphatase (TCdeltaC11 PTP) was determined in the range 1100-36,500 U/L by the fluorometric assay and 36-7100 U/L by the time-resolved immunofluorometric assay. The two nonisotopic assays should prove beneficial for the determination and study of various PTP.

References

Feb 1, 1975·Analytical Biochemistry·H H Hess, J E Derr
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Sep 14, 1992·Biochimica Et Biophysica Acta·D L Brautigan
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Citations

Jul 5, 2016·International Journal of Analytical Chemistry·Jun SumaokaMakoto Komiyama
Dec 12, 2007·Organic Letters·Matthew S TremblayDalibor Sames

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