规格型号:
(141.6 mL of reagent [R1 + R2] per kit;
equivalent to 456 reactions of 0.31 mL)
INTRODUCTION:
The most widely used method for enzymatic quantification of acetic acid is that based on the use of acetyl-coenzyme A synthetase (ACS), according to equations 1 – 3 below. For auto-analyser applications it becomes necessary to prepare a “master mix” reagent (R1), containing all components of the assay except the reaction initiation enzyme, ACS. However, reagent R1 when prepared from some kits has very limited on-machine stability, owing to a rapidly increasing absorbance value. To overcome this issue, Megazyme developed this kit (K-ACETAF), that doesn’t exhibit this instability phenomenon. Additionally, the anti-inhibitory compound polyvinylpyrollidone (PVP) has also been incorporated into the assay to prevent inhibition caused by certain tannins found in grape juice and wine.
KITS:
Kits suitable for the preparation of 141.6 mL of reagent (equivalent to 456 reactions of 0.31 mL) are available from Megazyme.
The kits contain the full assay method plus:
Bottle 1: Buffer (30 mL, pH 8.4) plus L-malic acid, PVP and sodium azide (0.02 % w/v) as a preservative. Stable for > 2 years at 4°C.
Bottle 2: (x2) NAD+ plus ATP and CoA.Freeze dried powder.Stable for > 5 years at -20°C.
Bottle 3: L-Malate dehydrogenase plus citrate synthase suspension, 2.2 mL. Stable for > 2 years at 4°C.
Bottle 4: Acetyl-coenzyme A synthetase suspension (1.1 mL). Stable for > 2 years at 4°C.
REAGENT PREPARATION:
Preparation of R1:
Component Volume
bottle 1 (buffer) 5.50 mL
bottle 2 (NAD+/ATP/CoA) 2.20 mL (after adding 5.50 mL of H2O to bottle 2)
bottle 3 (L-MDH/CS) 0.44 mL (swirl to mix before use)
H2O 18.35 mL
Total volume 26.49 mL
Preparation of R2:
Component Volume
bottle 4 (ACS) 0.22 mL (swirl to mix before use)
H2O 1.60 mL
Total volume 1.82 mL
EXAMPLE METHOD:
R1: 0.290 mL
Sample: ~ 0.005 mL
R2: 0.020 mL
Reaction time: 15 min at either 20-25°C or 37°C
Wavelength: 340 nm
Prepared reagent stability: > 3 days when refrigerated
Calculation: endpoint
Reaction direction: increase
Linearity: up to 30 μg/mL of acetic acid in final reaction solution