DESCRIPTION

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. BioAssay Systems' assay uses specific enzymecoupled 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 20 μL samples. Linear detection range in 96-well plate: 10 to 1000 μM galactose for colorimetric assays and 10 to 100 μM for fluorimetric assays.

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: 10 mL Enzyme Mix: 120 μL Dye Reagent: 120 μL Standard: 1 mL 10 mM Galactose Storage 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.

COLORIEMTRIC 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 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 Enzyme Mix in a refrigerator or on ice.

2. Standards and samples: prepare 400 μL 1000 μM Standard by mixing 40 μL 10 mM standard with 360 μL dH2O. Dilute standard in dH2O as follows.

3. Read fluorescence at lex = 530nm and lem = 585nm.

Notes: If the calculated galactose concentration of a sample is higher than 1000 μM in colorimetric assay or 100 μM in fluorimetric assay, dilute sample in water and repeat the assay. Multiply result by the dilution factor n.

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,

3. Gabrielli M. (1978). Serum galactose determination with centrifugal analyzers. Clin. Chem. 24:1990-1995.

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.

Assays of lactose in milk and other biological samples.

Assay Buffer: 10 mL Enzyme Mix: 120 μL Lactase: 120 μL

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 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).

2. Standards and samples: prepare 400 μL 2000 μM Standard by mixing 40 μL 20 mM standard with 360 μL dH2O. Dilute standard in dH2O as follows.

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.

For fluorimetric assays, the linear detection range is 6 to 100 μM lactose. Prepare 100 μM lactose standard by mixing 5 μL 20 mM standard with 995 μL H2O. Then dilute standards in H2O (see Colorimetric Procedure) to 100, 80, 60, 40, 30, 20, 10 and 0 μM.

1. Transfer 20 μL standards and 20 μL samples into separate wells of a black 96-well plate. Prepare Sample Blank if necessary.

3. Read fluorescence at lex = 530nm and lem = 585nm.

Notes: If the calculated lactose concentration of a sample is higher than 2000 μM in colorimetric assay or 100 μM in fluorimetric assay, dilute sample in water and repeat the assay. Multiply result  by the dilution factor n.

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,

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.

3. Tsenkova R, et al (1999). Near-infrared spectroscopy for dairy management: measurement of unhomogenized milk composition. J Dairy Sci. 82(11): 2344-2351.