Journal
TALANTA
Volume 73, Issue 3, Pages 471-475Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.talanta.2007.04.017
Keywords
ascorbic acid 2-phosphate; alkaline phosphatase; acid phosphatase; immunostaining; tetrazolium salt
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A new method to assay alkaline and acid phosphatases assay using ascorbic acid 2-phosphate (AsA-P) and ditetrazolium salt nitroblue tetrazolium chloride (NBT) was developed. AsA-P is hydrolyzed in the presence of phosphatase to yield ascorbic acid. In turn, the ascorbic acid reduces NBT directly or indirectly, opening the tetrazole ring to produce an insoluble formazan as a colored precipitate. The proposed method for alkaline phosphatase was compared with a conventional method in which 5-bromo-4-chloro-3-indolyl phosphate (BCIP) is used in combination with NBT in the dot blots of a dilution series of beta-lactoglobulin. AsA-P reduced NBT more effectively than BCIP in the presence of alkaline phosphatase. AsA-P could be also used as the chromogenic substrate for an acid phosphatase assay in the presence of phenazinium, methylsulfate and NBT. (C) 2007 Elsevier B.V. All rights reserved.
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