- A. MANUAL ASSAY PROCEDURE:
- B. AUTO-ANALYSER ASSAY PROCEDURE:
- C. MICROPLATE ASSAY PROCEDURE:
- SAMPLE PREPARATION:
1. Sample dilution.
The amount of D-lactic acid present in the cuvette (i.e. in the 0.1 mL of sample being analysed) should range between 0.5 and 30 μg. The sample solution must therefore be diluted sufficiently to yield a D-lactic acid concentration between 0.005 and 0.30 g/L.
2. Sample clarification.
a. Solutions:
Carrez I solution. Dissolve 3.60 g of potassium hexacyanoferrate (II) {K4[Fe(CN)6].3H2O} (Sigma cat. no. P9387) in 100 mL of distilled water. Store at room temperature.
Carrez II solution. Dissolve 7.20 g of zinc sulphate (ZnSO4.7H2O) (Sigma cat. no. Z4750) in 100 mL of distilled water. Store at room temperature.
Sodium hydroxide (NaOH, 100 mM). Dissolve 4 g of NaOH in 1 L of distilled water. Store at room temperature.
b. Procedure:
Pipette the liquid sample into a 100 mL volumetric flask which contains approx. 60 mL of distilled water, or weigh sufficient quantity of the sample into a 100 mL volumetric flask and add 60 mL of distilled water. Carefully add 5 mL of Carrez I solution, 5 mL of Carrez II solution and 10 mL of NaOH solution (100 mM). Mix after each addition. Fill the volumetric flask to the mark, mix and filter.
3. General considerations.
(a) Liquid samples: use clear, slightly coloured liquid samples directly in the assay.
(b) Acidic samples: if > 0.1 mL of an acidic sample is to be used undiluted (such as wine or fruit juice), the pH of the solution should be increased to approx. 10.0 using 2 M NaOH, and the solution incubated at room temperature for 30 min.
(c) Carbon dioxide: samples containing a significant amount of carbon dioxide, such as beer, should be degassed by increasing the pH to approx. 10.0 with 2 M NaOH and gentle stirring, or by stirring with a glass rod.
(d) Coloured samples: an additional sample blank, i.e. sample with no D-LDH, may be necessary in the case of coloured samples.
(e) Strongly coloured samples: if used undiluted, strongly coloured samples should be treated by the addition of 0.2 g of polyvinylpolypyrrolidone (PVPP) per 10 mL of sample. Shake the tube vigorously for 5 min and then filter through Whatman No. 1 filter paper.
(f) Solid samples: homogenise or crush solid samples in distilled water and filter if necessary.
(g) Samples containing fat: extract such samples with hot water at a temperature above the melting point of the fat, e.g. in a 100 mL volumetric flask at 60°C. Allow to cool to 20°C and fill the volumetric flask to the mark with distilled water. Store on ice or in a refrigerator for 15-30 min to allow the fat to separate and then filter. Discard the first few mL of filtrate and use the clear supernatant (which may be slightly opalescent) for assay. Alternatively, clarify with Carrez reagents.
(h) Samples containing protein: deproteinise samples containing protein by adding an equal volume of ice-cold 1 M perchloric acid with mixing. Centrifuge at 1,500 g for 10 min and neutralise the supernatant with 1 M KOH. Alternatively, use Carrez reagents.
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50 assays (manual) / 500 assays (microplate) / 450 assays (auto-analyser)
Bottle 1:
Buffer (25 mL, pH 10.0) plus D-glutamate and sodium azide (0.02% w/v) as a preservative.
Stable for > 2 years at 4°C.
Bottle 2:
NAD+.
Stable for > 5 years below -10°C.
Bottle 3:
D-Glutamate-pyruvate transaminase suspension (1.1 mL).
Stable for > 2 years at 4°C.
Bottle 4:
D-Lactate dehydrogenase suspension (1.1 mL).
Stable for > 2 years at 4°C.
Bottle 5:
D-Lactic acid standard solution (5 mL, 0.15 mg/mL) in 0.02% (w/v) sodium azide.
Stable for > 2 years; store sealed at 4°C.