- A. MANUAL ASSAY PROCEDURE:
- B. AUTO-ANALYSER ASSAY PROCEDURE:
This kit is suitable for the preparation of 132 mL of reagent (equivalent to 600 reactions of 0.230 mL). Reagent preparation is performed as follows:
- C. MICROPLATE ASSAY PROCEDURE:
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- SAMPLE PREPARATION:
1. Sample dilution.
The amount of glycerol present in the cuvette (i.e. in the 0.1 mL of sample being analysed) should range between 1.0 and 30 μg. The sample solution must therefore be diluted sufficiently to yield a concentration between 0.01 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: clear, slightly coloured and approximately neutral, liquid samples can be used 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. 7.4 using 2 M NaOH, and the solution incubated at room temperature for 30 min.
(c) Carbon dioxide: samples containing significant quantities of carbon dioxide, such as beer, should be degassed by increasing the pH to approx. 7.4 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 GK, 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)/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. Adjust to room temperature and fill the volumetric flask to the mark with water. Store on ice or in a refrigerator for 15-30 min 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 with Carrez reagents.
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70 assays (manual) / 700 assays (microplate) / 600 assays (auto-analyser)
Bottle 1:
Buffer (20 mL, pH 7.4).
Stable for > 2 years at 4°C.
Bottle 2: *2
NAD+, ATP and D-glucose. Freeze dried powder.
Stable for > 5 years below -10°C.
Bottle 3:
ADP-glucokinase plus glucose-6-phosphate dehydrogenase suspension, 1.5 mL.
Stable for > 2 years at 4°C.
Bottle 4:
Glycerokinase suspension (1.5 mL).
Stable for > 2 years at 4°C.
Bottle 5:
Glycerol standard solution (5 mL, 0.20 mg/mL) in 0.02% (w/v) sodium azide.
Stable for > 2 years at 4°C.
