Amplite® Lactose Quantitation Kit

Thaw all the kit components to room temperature before starting the experiment.

1. Prepare and add Lactose Standards and test samples (50 µL)

2. Prepare and add Enzyme-dye assay reaction mixture (50 µL)

3. Incubate at 37 °C for 10 - 30 minutes

4. Monitor Fluorescence intensity at Ex/Em = 540/590 nm or absorbance at OD = 570 nm

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

1. Prepare stock solution by adding 25 μL of DMSO (Component E) into the vial of Amplite® Red (Component A). The stock solution should be used promptly. Any remaining solution should be aliquoted and refrozen at -20 °C.

   Note: Amplite® Red is unstable in the presence of thiols such as dithiothreitol (DTT) and 2-mercaptoethanol. The final concentration of DTT or 2-mercaptoethanol in the reaction should be no higher than 10 µM.

   Note: Amplite® Red is unstable at high pH (>8.5). The reaction should be performed at pH 7 – 8. The provided assay buffer (pH 7.4) is recommended.

1. Add 1 mL of assay buffer (Component B) into the vial of horseradish peroxidase (Component C).

   Note: The unused HRP solution should be divided into single-use aliquots and stored at -20 °C.

1. Add 500 µL of assay buffer (Component B) into the vial of Lactose Standard (Component F) to make a 20 mM stock solution. Diluted to 1 mM in assay buffer (10 uL of 20 mM to 190 uL assay buffer).

   Note: The unused Lactose Standard stock solution should be stored at -20 °C.

1. Add 100 µL of assay buffer (Component B) into the vial of enzyme mix (Component D).

   Note: The unused enzyme mix stock solution should be divided into single-use aliquots and stored at -20 °C.

Prepare the enzyme-dye assay reaction mixture according to the following table, protected from light.

LS=Sodium Standards (LS1 - LS7, 200 to 3.12 µM for colorimetric analysis and 50 to 0.78 µM for fluorometric analysis); BL=Blank Control; TS=Test Samples

1. Prepare Lactose Standards (LS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

2. Add 50 µL of Enzyme-dye reaction mixture to each well of Sodium Standards, blank control, and test samples to make the assay volume 100 µL/well. For a 384-well plate, add 25 µL into each well instead, for a total volume of 50 µL/well.

3. Incubate the reaction at room temperature for 5 to 10 minutes, protected from light.

4. Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 540/590 nm (cut off at 570 nm) or absorbance with an absorbance microplate reader at OD = 570 nm.

Add 50 µL of a diluted Lactose Standard stock solution (1 mM) to 200 µL of Assay Buffer (Component B) to make a 200 µM (LS1) dilution. Then perform 1:2 serial dilutions to get serially diluted Standard (LS2 – LS7).