Enzymatic Method for Determining Galactose (Galactose Assay)

This assay protocol is suitable for the colorimetric/fluorometric detection of Galactose and Lactose in cell and tissue culture supernatants, urine, plasma, serum, and other biological samples using the Galactose Assay Kit (MAK012). In this assay kit, galactose is oxidized by galactose oxidase resulting in a colorimetric (570 nm)/ fluorometric (λex = 535 nm/λem = 587 nm) product, proportional to the galactose present. This kit has a linear detection range of 0.2–1.0 nmole galactose for the fluorometric assay and 2–10 nmoles galactose for the colorimetric assay. 

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. 

Reagents

Galactose Assay Buffer                                         25 mL
    Catalog Number MAK012A
 
Galactose Probe, in DMSO                                   0.2 mL
    Catalog Number MAK012B
 
Galactose Enzyme Mix                                           1 vl
    Catalog Number MAK012C
 
Horseradish Peroxidase (HRP)                                 1 vl
    Catalog Number MAK012D
 
Galactose Standard, 100 nmole/μL                        0.1 mL
    Catalog Number MAK012E

Reagents and Equipment Required but Not Provided

96 well flat-bottom plate – It is recommended to use black plates with clear bottoms (Catalog Number M5686 or equivalent) for fluorescence assays and clear plates (Catalog Number M4436 or equivalent) for colorimetric assays.

Fluorescence or spectrophotometric multiwell plate reader

Preparation Instructions

Galactose Assay Buffer – Allow buffer to come to room temperature before use.

Galactose Probe – Ready-to-use as supplied. Allow Galactose Probe to come to room temperature before use. Store protected from light at –20 °C for use within 2 months.

For the fluorescence assay, dilute an aliquot of the colorimetric Galactose Probe Solution 5 to 10-fold with Galactose Assay Buffer just prior to use. This will reduce the background of the fluorescence assay.

Galactose Enzyme Mix and Horseradish Peroxidase – Reconstitute each in 220 μL of Galactose Assay Buffer. Mix well by pipetting, then aliquot each and store protected from light at –20 °C. Use within
2 months of reconstitution and keep cold while in use.

Storage/Stability

The kit is shipped on wet ice. Storage at –20 °C, protected from light, is recommended.

All samples and standards should be run in duplicate.
 
Galactose Standards for Colorimetric Detection
Dilute 10 μL of the 100 mM (100 nmole/μL) Galactose Standard Solution with 990 μL of Galactose Assay Buffer to prepare a 1 mM (1 nmole/μL) standard solution. Mix well and add 0, 2, 4, 6, 8, and 10 μL of the 1 mM Galactose standard solution into a 96 well plate, generating 0 (assay blank), 2, 4, 6, 8, and 10 nmole/well standards. Add Galactose Assay Buffer to each well to bring the volume to 50 μL.
 
Galactose Standards for Fluorometric Detection
Prepare a 1 mM Galactose standard solutions as for the colorimetric assay. Dilute 20 μL of the 1mM standard solution with 180 μL of Galactose Assay Buffer to generate a 0.1 mM (0.1 nmole/μL) standard solution. Add 0, 2, 4, 6, 8, and 10 μL of the 0.1 mM Galactose standard solution into a 96 well plate, generating 0 (assay blank), 0.2, 0.4, 0.6, 0.8, and 1.0 nmole/well standards. Add Galactose Assay Buffer to each well to bring the volume to 50 μL.
 
Sample Preparation Liquid samples can be measured directly. Bring samples to a final volume of 50 μL with Galactose Assay Buffer.
 
For unknown samples, it is suggested to test several sample dilutions to ensure the readings are within the linear range of the standard curve.
 
Assay Reaction
1.     Set up the Master Reaction Mix according to the scheme in Table 1. 50 μL of the Master Reaction Mix is required for each reaction (well).

Table 1.
Reaction Mixes

Reagent Volume
Galactose Assay Buffer 44 μL
Galactose Probe 2 μL
Galactose Enzyme Mix 2 μL
HRP 2 μL

2.     Add 50 μL of the Master Reaction Mix to each of the wells. Mix well using a horizontal shaker or by pipetting, and incubate the reaction for 60 minutes at 37 °C. Protect the plate from light during the incubation.

3.     For colorimetric assays, measure the absorbance at 570 nm (A570). For fluorometric assays, measure fluorescence intensity (λex = 535/λem = 590 nm).

Results

Calculations
The background for the assays is the value obtained for the 0 (assay blank) Galactose Standard. Correct for the background by subtracting the 0 (assay blank) value from all readings. Background values can be significant and must be subtracted from all readings. Use the values obtained from the appropriate Galactose standards to plot a standard curve.
Note: A new standard curve must be set up each time the assay is run.

Concentration of Galactose

Sa/Sv = C



Sa = Amount of Galactose in unknown sample (nmole)
from standard curve
Sv = Sample volume (μL) added into the wells
C = Concentration of Galactose in sample

Sample Calculation
Amount of Galactose (Sa) = 5.84 nmole
(from standard curve)
Sample volume (Sv) = 50 μL

Molecular weight of galactose = 180.16

Concentration of Galactose in sample

5.84 nmole/50 μL = 0.1168 nmole/μL

0.1168 nmole/μL x 180.16 ng/nmole = 21.04 ng/μL

 

Materials

     
Related Links

Categories