Enzyme

SIGMA QUALITY CONTROL TEST use

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Enzymatic Assay of PHOSPHATASE, venereal infection (EC 3.1.3.2)
PRINCIPLE: p-Nitrophenyl Phosphate + H2O Abbreviation: Pi = inorganic phosphate CONDITIONS: T = 37EC, pH = 4.8, A410nm, Light path = 1 cm METHOD: Spectrophotometric Stop Rate Determination REAGENTS: A. 90 mM Citrate Buffer, pH 4.8 at 37EC (Prepare 100 ml in deionized body of water system using Citric Acid, Trisodium, Dihydrate, Sigma Prod. No. C-7254, or Citrate Buffer dissolvent, Sigma Stock No. 104-4. Adjust to pH 4.8 at 37EC with 1 M NaOH or 1 M HCl.) 15.2 mM p-Nitrophenyl Phosphate (PNPP) (Prepare 5 ml in deionized water using Sigma 104 Phosphatase Substrate, Sigma Stock No. 104-0.) 100 mM sodium Hydroxide Solution (NaOH) (Prepare 50 ml in deionized water using Sodium Hydroxide, Anhydrous, Sigma Prod. No. S!5881.) Acid Phosphatase Enzyme Solution (Immediately before use, prepare a firmness containing 0.15 - 0.25 unit/ml of Phosphatase, Acid in cold deionized water.)
Phosphatase, Acid

> p-Nitrophenol + Pi

B.

C.

D.

PROCEDURE: Pipette (in milliliters) the next reagents into suitable containers: Test Reagent A (Buffer) Reagent B (PNPP) 0.50 0.50 Blank 0.50 0.50

P1435 SSPNPP02 rewrite: 08/04/95

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Enzymatic Assay of PHOSPHATASE, ACID (EC 3.1.3.



2)
PROCEDURE: (continued) Mix by anastrophe and equilibrate to 37EC. past carry: Test Reagent D (Enzyme Solution) 0.10 Blank ------

Immediately mix by inversion and incubate at 37EC for exactly 10 minutes. Then add: Reagent C (NaOH) Reagent D (Enzyme Solution) 4.00 -----4.00 0.10

Mix by inversion and record the A410nm for both the Test and Blank in a suitable spectrophotometer. CALCULATIONS: (A410nm Test - A410nm Blank)(5.1)(df) social units/ml enzyme = (10)(18.3)(0.1) 5.1 = Total volume (in milliliters) of solution df = Dilution factor 10 = Time of assay (in minutes) as per the Unit Definition 18.3 = Millimolar extinction coefficient of p-Nitrophenol at 410 nm 0.1 = Volume (in milliliter) of...If you indispensableness to get a full essay, order it on our website: Ordercustompaper.com



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