A sensitive, high-volume, colorimetric assay for protein phosphatases.
Protein phosphatases are intimately involved in a variety of cellular processes, many of which are of interest to the pharmaceutical industry. Phosphatase assays generally employ radioisotopes, making them tedious to perform, costly, and hazardous, while other procedures require antibodies and/or are unsuitable for mass screening efforts. To facilitate screening for inhibitors of the CD45 protein tyrosine phosphatase (PTPase), we have developed a sensitive colorimetric assay, using small volumes in 96-well microtiter plates and read on a standard ELISA plate reader. The assay was sensitive down to 0.5 nmol of released phosphate and can be easily run by robotics to assay thousands of compounds in a day. The assay is sparing of reagents and has been successfully used with all classes of phosphatases. The reagents are nonradioactive, readily obtainable, and minimal in cost. This assay should facilitate the search for specific inhibitors of phosphatases.
Fisher
D K
DK
Department of Immunopharmacology, Sterling Winthrop Pharmaceuticals Research Division, Collegeville, Pennsylvania 19426-0900.
Higgins
T J
TJ
D000595:Amino Acid Sequence; D003124:Colorimetry; D004396:Coloring Agents; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D010920:Placenta / Q000201:enzymology; D010936:Plant Extracts / Q000494:pharmacology; D011247:Pregnancy; D017027:Protein Tyrosine Phosphatases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D012394:Rosaniline Dyes; D013056:Spectrophotometry, Ultraviolet
D004396:Coloring Agents; D010936:Plant Extracts; D012394:Rosaniline Dyes; C005095:malachite green; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases