Lactose (C12H22O11), also called milk sugar, is a disaccharide that consists of b-D-galactose and a/b-D-glucose through a b1-4 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:. Lactose Assay Kit 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.
Key Features:Use as little as 20ul samples. Linear detection range in 96-well plate: 17 to 2000uM lactose for Colorimetric Assay:s and 6 to 100uM for Fluorometric Assays.
Applications: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.
Kit Contents:Assay Buffer: 10ml Enzyme Mix: 120ul Lactase: 120ulDye Reagent: 120ul Standard: 1ml 20mM LactoseStorage conditions. The kit is shipped on dry ice. Store all components at -20°C. Shelf life of three months after receipt
Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.
Colorimetric Procedure:Note: (1) glycerol and SH-containing reagents (e.g. b–mercaptoethanol, 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 600ul milk with 100ul 6 N HCl. Centrifuge 5 min at 14,000 rpm. Transfer 300ul supernatant into a clean tube and neutralize with 50ul 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 400ul 2000uM Standard by mixing 40ul 20mM standard with 360ul dH2O. Dilute standard in dH2O as follows
No 2000uM STD+H2O Vol (ul) Lactose (uM)1 100ul+0ul 100 20002 80ul+20ul 100 16003 60ul+40ul 100 12004 40ul+60ul 100 8005 30ul+70ul 100 6006 20ul+80ul 100 4007 10ul+90ul 100 2008 0ul +100ul 100 0Transfer 20ul standards and 20ul samples into separate wells of a clear flat-bottom 96-well plate. Note: if a sample is known to contain galactose, transfer 20ul sample in duplicate (one sample and one sample blank)
3. Reaction. For each reaction well, mix 85ul Assay Buffer, 1ul Lactase, 1ul Enzyme Mix (vortex briefly before pipetting), and 1ul Dye Reagent in a clean tube. (Note: for the sample blanks, prepare a control Working Reagent which is the same except WITHOUT the 1ul Lactase). Transfer 80ul 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).
Fluorometric Procedure:For Fluorometric Assay:s, the linear detection range is 6 to 100uM lactose. Prepare 100uM lactose standard by mixing 5ul 20mM standard with 995ul H2O. Then dilute standards in H2O (see Colorimetric Procedure:) to 100, 80, 60, 40, 30, 20, 10 and 0uM
1. Transfer 20ul standards and 20ul samples into separate wells of a black 96-well plate. Prepare Sample Blank if necessary
2. Add 80ul Working Reagent, tap plate to mix. Incubate 30 min
3. Read fluorescence at lex=530nm and lem=585nm
Notes: If the calculated lactose concentration of a sample is higher than 2000uM in Colorimetric Assay: or 100uM in Fluorometric Assay:, dilute sample in water and repeat the assay. Multiply result by the dilution factor n.
Calculation:Subtract blank value (water, #8) from the standard values and plot the DOD or DRFU against standard concentrations. Determine the slope and calculate the lactose concentration of Sample, [Lactose]=ODSAMPLE–ODBLANKSlope× n (uM)[Lactose] =RFUSAMPLE–RFUBLANKSlope× n (uM)Colorimetry:Fluorimetry:ODSAMPLE, ODBLANK, RFUSAMPLE, RFUBLANK are optical density and fluorescence values of the Sample and Blank. The Blank is water if there is no galactose, and Sample Blank if sample contains galactose. n is the dilution factor
Conversions: 1mM lactose equals 36 mg/dL, 0.036% or 360 ppm.
Materials Required, But Not Provided:Pipetting devices, centrifuge tubes, clear flat-bottom 96-well plates, optical density plate reader; black 96-well plates and fluorescence plate reader.
