- A. MANUAL ASSAY PROCEDURE:
- B. AUTO-ANALYSER ASSAY PROCEDURE:
- C. MICROPLATE ASSAY PROCEDURE:
- SAMPLE PREPARATION:
1. Sample dilution.
The amount of succinic acid present in the cuvette (i.e. in the 0.1 mL of sample being analysed) should range between 0.8 and 40 μg. The sample solution must therefore be diluted sufficiently to yield a succinic acid concentration between 0.008 and 0.40 g/L.
2. 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. 8.4 using 2 M NaOH, and the solution incubated at room temperature for 30 min.
(c) Carbon dioxide: samples containing significant amounts of carbon dioxide, such as beer, should be degassed by increasing the pH to approx. 8.4 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 SCS, 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. Adjust to room temperature and fill the volumetric flask to the mark with distilled 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 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 K-CARREZ.
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20 assays (manual) / 200 assays (microplate) / 270 assays (auto-analyser)
Bottle 1:
Buffer (8 mL, pH 8.4) plus sodium azide (0.02% w/v) as a preservative.
Store at 4°C. See individual label for expiry.
Bottle 2: *2
NADH plus stabiliser.
Store below -10°C. See individual label for expiry.
Bottle 3: *2
ATP plus PEP and CoA.
Store below -10°C. See individual label for expiry.
Bottle 4:
Pyruvate kinase plus lactate dehydrogenase suspension, 0.55 mL.
Store at 4°C. See individual label for expiry.
Bottle 5:
Succinyl-CoA synthetase suspension (0.55 mL).
Store at 4°C. See individual label for expiry.
Bottle 6:
Succinic acid (~ 2 g).
Store at 4°C. See individual label for expiry.
