(1 reference)-
- Target
- Lactose
- Application
- Biochemical Assay (BCA)
- Sample Type
- Beverages, Food, Milk
- Specificity
- 6 μM
- Characteristics
- Use as little as 20 µL samples. Linear detection range in 96-well plate: 17 to 2000 µM lactose for colorimetric assays and 6 to 100 µM for fluorimetric assays.
- Components
- Assay Buffer: 10 mL. Enzyme Mix: 120 µL. Lactase: 120 µL. Dye Reagent: 120 µL. Standard: 1 mL 20 mM Lactose.
- Material not included
- Pipetting devices, centrifuge tubes, clear flat-bottom 96-well plates, optical density plate reader, black 96-well plates and fluorescence plate reader.
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- Application Notes
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Assays of lactose in milk and other biological samples.
Drug Discovery/Pharmacology: effects of drugs on lactose metabolism.
Food and Beverages: lactose in food and beverages products. - Protocol
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Note: (1) glycerol and SH-containing reagents (e.g. µMercaptoethanol, dithiothreitol) are known to interfere in this assay and should be avoided in sample preparation. (2) For samples containing galactose, a sample blank is necessary (see Procedure), (3) This assay is based on a kinetic reaction. To ensure identical incubation time, addition of Working Reagent to standard and samples should be quick and mixing should be brief but thorough. Use of a multi-channel pipettor is recommended. Sample treatment: Milk samples should be cleared by mixing 600 µL milk with 100 µL 6 N HCl. Centrifuge 5 min at 14,000 rpm. Transfer 300 µL supernatant into a clean tube and neutralize with 50 µL 6 N NaOH. The neutralized supernatant is ready for assay (dilution factor n = 1.36).
1. Equilibrate all components to room temperature. During experiment, keep thawed Lactase and Enzyme Mix in a refrigerator or on ice.
2. Standards and samples: prepare 400 µL 2000 µMStandard by mixing 40 µL 20 mM standard with 360 µL dH2O. Dilute standard in dH2O. Transfer 20 µL standards and 20 µL samples into separate wells of a clear flat-bottom 96-well plate. Note: if a sample is known to contain galactose, transfer 20 µL sample in duplicate (one sample and one sample blank).
3. Reaction. For each reaction well, mix 85 µL Assay Buffer, 1 µL Lactase, 1 µL Enzyme Mix (vortex briefly before pipetting), and 1 µL Dye Reagent in a clean tube. (Note: for the sample blanks, prepare a control Working Reagent which is the same except WITHOUT the 1 µL Lactase). Transfer 80 µL Working Reagent into each reaction (and control) well. Tap plate to mix. Incubate 30 min at room temperature.
4. Read optical density at 570nm (550-585nm). - Calculation of Results
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Subtract blank value (water, #8) from the standard values and plot the OD or RFU against standard concentrations.
Conversions: 1 mM lactose equals 36 mg/dL, 0.036% or 360 ppm. - Restrictions
- For Research Use only
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- Storage
- -20 °C
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: "Dietary protein restriction induces steatohepatitis and alters leptin/signal transducers and activators of transcription 3 signaling in lactating rats." in: The Journal of nutritional biochemistry, Vol. 23, Issue 7, pp. 791-9, (2012) (PubMed).
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: "Dietary protein restriction induces steatohepatitis and alters leptin/signal transducers and activators of transcription 3 signaling in lactating rats." in: The Journal of nutritional biochemistry, Vol. 23, Issue 7, pp. 791-9, (2012) (PubMed).
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- Target
- Lactose
- Synonyms
- LAC Kit, LPH Kit, LPH1 Kit, lactase Kit, LCT Kit
- Target Type
- Chemical
- Background
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Quantitative determination of lactose by colorimetric (570nm) or fluorimetric (530nm/590nm) methods.
Procedure: 30 min.
Lactose (C12H22O11), also called milk sugar, is a disaccharide that consists of beta-D-galactose and alpha/beta-D-glucose through a 1-4 glycosidic linkage. Lactose is the major sugar and makes up 2-8% of milk. Simple, direct and high-throughput assays for lactose determination find wide applications. This assay uses specific enzyme-coupled reactions in which lactose is cleaved and the resulting galactose forms a colored product. The color intensity at 570nm or fluorescence intensity at 530nm/585nm is directly proportional to the lactose concentration in the sample.
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