Galactose Assay Kit

Galactose Assay Kit

(Catalog #K621-100; 100 assays; Store at -20^oC)

I. Introduction:

Galactose (C₆H₁₂O₆ FW: 180.16) is one the major naturally occurring sugars, involved in many biological processes. BioVision’s Galactose Assay Kit provides a simple, convenient means for direct measurement of galactose levels in various biological samples (serum, plasma, other body fluids, food, growth media, etc.). In the assay kit, Galactose was specifically oxidized generating a product that reacts with the Galactose Probe to produce color (O.D 570 nm) and fluorescence (Ex/Em 535/587 nm). Therefore, galactose can be easily quantified. Liquid samples can be tested directly without purification. The assay is fast, convenient and sensitive. It can also be used in high throughput assay.

1. 
   

   Kit Contents

   
   

Components	K621-100	Cap Code	Part No.
Galactose Assay Buffer Galactose Probe (Lyophilized) Dimethylsulfoxide (DMSO, Anhydrous) Galactose Enzyme Mix (Lyophilized) Galactose Standard (100 nmol/ml)	25 ml 1 Vial 0.4 ml 1 Vial 100 ml	WM Red Brown Green Yellow	K621-100-1 K621-100-2 K621-100-3 K621-100-4 K621-100-5

2. 
   

   Storage and Handling:

   
   

Store kit at –20^oC, protect from light. Briefly centrifuge vials prior to opening. Allow buffer warm to room temperature before use, but keep enzymes on ice during the assay.

3. 
   

   Reagent Preparation:

   
   

Galactose Probe: Dissolve in 220 μl DMSO (provided) before use. Store at –20^oC, protect from light and moisture. Use within two months.

Galactose Enzyme Mix: Dissolve in 220 μl Galactose Assay Buffer. Aliquot and store at –20^oC. Use within two months.

4. 
   

   Galactose Assay Protocol:

   
   

1. Standard Curve Preparation:

For colorimetric assay, dilute the Galactose Standard to 1 nmol/μl by adding 10 μl of the 100 nmol/μl Galactose Standard to 990 μl of Galactose Assay Buffer and mix well. Add 0, 2, 4, 6, 8, 10 μl into a series of wells. Adjust the volume to 50 μl/well with Galactose Assay Buffer to generate 0, 2, 4, 6, 8, 10 nmol/well of Galactose Standard.

For fluorometric assay, dilute the Galactose Standard solution to 0.1 nmol/μl by adding 10 μl of the Galactose Standard to 990 μl of Galactose Assay Buffer and mix well. Then take 20 μl into 180 μl of Galactose Assay Buffer and mix well. Add 0, 2, 4, 6, 8, 10 μl into each well individually. Adjust volume to 50 μl/well with Galactose Assay Buffer to generate 0, 0.2, 0.4, 0.6, 0.8, 1.0 nmol/well of the Galactose Standard.

1. 
   

   Sample Preparation: Different amounts of sample can be directly added into the plate, then adjust to total volume to 50 μl with Galactose Assay Buffer. For unknown samples, we suggest testing several doses of sample to ensure the readings are within the standard curve linear range.

   
   


2. 
   

   Galactose Reaction Mix: Mix enough reagent for the number of assays to be performed. For each well, prepare a total 50 ml Reaction Mix containing:

   
   

 

 

 

46 μl Galactose Assay Buffer

2 μl Galactose Probe*

2 μl Galactose Enzyme Mix

*Note: The fluorometric assay is ~10 fold more sensitive than the colorimetric assay. Use 0.4 μl probe in each reaction in the fluorometric assay will decrease background fluorescence reading significantly.

3. 
   

   Mix well. Add 50 μl of the Reaction Mix to each well containing the Galactose Standard and test samples. Mix well.

   
   


4. 
   

   Incubate the reaction for 30 minutes at 37^oC, protect from light.

   
   


5. 
   

   Measure O.D. 570nm for the colorimetric assay or Ex/Em = 535/590 nm for the fluorometric assay in a microplate reader.

   
   


6. 
   

   Calculations: Correct background by subtracting the value of the 0 galactose control from all sample readings. Plot standard curve galactose amount (nmol) vs OD 570nm. Apply sample readings to the standard curve. Galactose concentration:

   
   

C = Ga/Sv nmol/ml or mM

Where Ga: Galactose amount in the sample wells (in nmol).

Sv: Sample volume added into the sample wells (in μl).

Galactose Molecular Weight: 180.16.