Galactose Assay Kit

Catalog No. ABIN1000309

Product Details

Target: Galactose

Application: Biochemical Assay (BCA)

Sample Type: Cell Culture Supernatant, Milk, Plasma, Saliva, Serum, Urine

Specificity: 10 μM

Characteristics: 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.

Components: Assay Buffer: 10 mL. Enzyme Mix: 120 µL. Dye Reagent: 120 µL. Standard: 1 mL 10 mM Galactose.

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.

Application Details

Application Notes: 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.

Protocol: 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) 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.
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 µMStandard by mixing 40 µL 10 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.
3. Reaction. For each reaction well, mix 85 µL Assay Buffer, 1 µL Enzyme Mix (vortex briefly before pipetting), and 1 µL Dye Reagent in a clean tube. Transfer 80 µL Working Reagent into each reaction well. Tap plate to mix. Incubate 20 min at room temperature.
4. Read optical density at 570nm (550-585nm).

Sample Preparation:

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)

Calculation of Results:

Subtract blank value (water, #8) from the standard values and plot the OD or RFU against standard concentrations.
Conversions: 1 mM galactose equals 18 mg/dL, 0.018% or 180 ppm.

Restrictions: For Research Use only

Handling

Storage: -20 °C

References

Sun, Zhao, Ye, Fan, Liu, Tan: "An investigation into the relationship between metabolic responses and energy regulation in antibody-producing cell." in: Journal of microbiology and biotechnology, Vol. 23, Issue 11, pp. 1586-97, (2013) (PubMed).

Target Details

Target: Galactose

Background: Quantitative determination of galactose by colorimetric (570nm) or fluorimetric (530nm/590nm) methods.
Procedure: 20 min.

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. This 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.