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

Abstract：

OBJECTIVE:To develop sensitive nonisotopic assays for protein-tyrosine phosphatase (PTP) activity. METHODS: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. RESULTS: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. CONCLUSIONS:The two nonisotopic assays should prove beneficial for the determination and study of various PTP.

journal_name

Clin Biochem

journal_title

Clinical biochemistry

authors

Galvan B,Christopoulos TK

doi

10.1016/0009-9120(95)02026-8

subject

Has Abstract

pub_date

1996-04-01 00:00:00

pages

125-31

issue

2

eissn

0009-9120

issn

1873-2933

pii

0009-9120(95)02026-8

journal_volume

29

pub_type

杂志文章

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