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 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 20 mL samples. Linear detection range in 96-well plate: 10 to 1000 mM galactose for colorimetric assays and 10 to 100 mM 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 mL       

Dye Reagent: 120 mL     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 mL milk with 100 mL 6 N HCl. Centrifuge 5 min at 14,000 rpm. Transfer 300 mL supernatant into a clean tube and neutralize with 50 mL 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 mL 1000 mM Standard by mixing 40 mL 10 mM standard with 360 mL dH₂O. Dilute standard in dH₂O as follows.

Transfer 20 mL standards and 20 mL samples into separate wells of a clear flat-bottom 96-well plate.

3. Reaction. For each reaction well, mix 85 mL Assay Buffer, 1 mL Enzyme Mix  (vortex briefly before pipetting), and  1 mL Dye Reagent in a

clean tube. Transfer 80 mL Working Reagent into each reaction well. Tap plate to mix. Incubate 20 min at room temperature.

4. Read optical density at 570nm (550-585nm).

Fluorimetric Procedure

For fluorimetric assays, the linear detection range is 10 to 100 mM galactose. Prepare 100 mM galactose standard by mixing 10 mL 10 mM standard with 990 mL H2O. Then dilute standards in H2O (see Colorimetric Procedure) to 100, 80, 60, 40, 30, 20, 10 and 0 mM.

1. Transfer 20 mL standards and 20 mL samples into separate wells of a black 96-well plate.

2. Add 80 mL Working Reagent, tap plate to mix. Incubate 20 min.

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