ALKALINE PHOSPHATASE Chemistry Reagents

ALKALINE PHOSPHATASE Chemistry Reagents Colorimetric method KINETIC PRINCIPLE Alkaline phosphatase (ALP) catalyze the hydrolysis of 4-nitrophenylphosphate (4-NPP) with the formation of free 4-nitrophenol and inorganic phosphate, acting the alkaline buffer as a phosphate-group acceptor. The reaction is monitored kinetically at 405 nm by the rate of formation of 4-nitrophenol, proportional to the activity of ALP present in the sample. REAGENT COMPOSITION 3 x 1oo ml R1 ? ALP buffer:?DEA buffer 1.25 mol/L pH 10.2, magnesium chloride 0.6 mmol/L. Biocides. 3 x 80 ml R2- ALP substrate: ?4-NPP 50 mmol/L. Biocides. 1 x 60 ml STORAGE AND STABILITY Store at at 2-8?C REAGENT PREPARATION Working reagent. Mix 4 mL of R1 + 1 mL of R2. Stable for 5 days at 20-25?C or 15-30 days at 2-8?C, depending on the remaining caducity of both reagents. Protect from light. SAMPLES Serum or heparinized plasma, free of hemolysis. Other anticoagulants such as EDTA, oxalate and citrate inhibit the enzyme by complexing Mg++ and should not be used. Alkaline phosphatase in serum or plasma is stable for 7 days at 2-8?C. MATERIALS REQUIRED Photometer or spectrophotometer with a thermostatic cell compartment set at 25/30/37?C, capable of reading at 405 nm. Stopwatch, strip-chart recorder or printer. Cuvettes with 1-cm pathlength. Pipettes to measure reagent and samples. ALKALINE PHOSPHATASE Chemistry Reagents per box