Galactose (C6H12O6) is a monosaccharide that is found in dairy products, sugar beets, gums and mucilages. It is also synthesized in mammals, where it forms part of glycolipids and glycoproteins in several tissues. It forms the disaccharide lactose when combined with glucose. Simple, direct and high-throughput assays for galactose determination find wide applications.
Galactose Assay Kit uses specific enzyme-coupled reactions to form a colored product. The color intensity at 570nm or fluorescence intensity at 530nm/585nm is directly proportional to the galactose concentration in the sample.
Key Features:Use as little as 20ul samples. Linear detection range in 96-well plate: 10 to 1000uM galactose for Colorimetric Assay:s and 10 to 100uM for Fluorometric Assay:s.
Applications:Direct Assays: galactose in serum, plasma, urine, saliva, milk, culture medium and other biological samples. Drug Discovery/Pharmacology: effects of drugs on galactose metabolism
Food and Beverages: galactose in food and beverages products.
Kit Contents:Assay Buffer: 10ml Enzyme Mix: 120ulDye Reagent: 120ul Standard: 1ml 10mM GalactoseStorage 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) 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: serum and plasma samples can be assayed directly. 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 Enzyme Mix in a refrigerator or on ice
2. Standards and samples: prepare 400ul 1000uM Standard by mixing 40ul 10mM standard with 360ul dH2O. Dilute standard in dH2O as follows
No 1000uM STD+H2O Vol (ul) Galactose (uM)1 100ul+0ul 100 10002 80ul+20ul 100 8003 60ul+40ul 100 6004 40ul+60ul 100 4005 30ul+70ul 100 3006 20ul+80ul 100 2007 10ul+90ul 100 1008 0ul +100ul 100 0Transfer 20ul standards and 20ul samples into separate wells of a clear flat-bottom 96-well plate
3. Reaction. For each reaction well, mix 85ul Assay Buffer, 1ul Enzyme Mix (vortex briefly before pipetting), and 1ul Dye Reagent in a clean tube. Transfer 80ul Working Reagent into each reaction well. Tap plate to mix. Incubate 20 min at room temperature
4. Read optical density at 570nm (550-585nm).
Fluorometric Procedure:For Fluorometric Assays, the linear detection range is 10 to 100uM galactose. Prepare 100uM galactose standard by mixing 10ul 10mM standard with 990ul 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
2. Add 80ul Working Reagent, tap plate to mix. Incubate 20 min
3. Read fluorescence at lex=530nm and lem=585nm
Notes: If the calculated galactose concentration of a sample is higher than 1000uM 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 galactose concentration of Sample,[Galactose] =ODSAMPLE–ODH2OSlope× n (uM)Colorimetry: [Galactose] =RFUSAMPLE–RFUH2OSlope× n (uM)Fluorimetry: ODSAMPLE, ODH2O are optical density values of the sample and water. RFUSAMPLE, RFUH2O are fluorescence intensity values of the sample and water. n is the dilution factor
Conversions: 1mM galactose equals 18 mg/dL, 0.018% or 180 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.
