Maltose Phosphorylase Activity Kit (Fluorometric)

Storage Conditions and Reagent Preparation:
Store the kit at -20°C. The kit components are stable for one year when stored as recommended. Briefly centrifuge small vials at low speed prior to opening. Read the entire protocol before performing the experiment.
 Maltose Assay Buffer: Ready to use as supplied. Warm bottle to room temperature (RT) before use. Store at 4°C.
 Maltose Probe (in DMSO): Ready to use as supplied. Warm to RT prior to use to melt frozen DMSO. Store at -20°C, protected from
light and moisture. Use within two months.
 Maltose Developer and Maltose: Reconstitute each component with 220 µl of Assay Buffer. Store at -20°C. Keep on ice while in use.
Use within two months.
 Glucose Standard: Store at -20°C. Warm to RT before use.
 Maltose Phosphorylase: Add 450 μl of 50% glycerol (not included) to the vial to prepare the enzyme stock. Vortex to mix. Aliquot and
store at -20°C. Avoid multiple freeze-thaw cycles. Use within two months.

VII. Maltose Phosphorylase Activity Assay Protocol:

1. Sample Preparation: Grow cells in a suitable growth medium at 37°C overnight. After incubation, centrifuge at 10,000 x g for 20 min to harvest the cells and measure the weight of the pellet. Add 5 ml of ice-cold PBS into the cell pellet and disperse the pellet. Sonicate the cells for 5-10 min on ice. After sonication, centrifuge the cells at 10,000 x g for 30 min and transfer the supernatant to a new tube. For Sample and Background wells, add 2 μl of the cell supernatant to the desired well(s) in a 96-well black flat-bottom plate. Adjust the volume to 50 μl using Maltose Assay Buffer.
For Positive Control, prepare a 10 fold dilution of the Maltose Phosphorylase enzyme by adding 5 μl of the enzyme with 45 μl of Maltose Assay Buffer. Add 2 μl of the diluted enzyme into the Positive Control well. Adjust the volume to 50 μl using Maltose Assay Buffer.
2. Standard Curve Preparation: Mix 10 µl of Glucose Standard with 990 µl of Assay Buffer to prepare 1 mM diluted Glucose Standard. Mix 100 µl of 1 mM diluted Glucose Standard with 900 µl of Assay Buffer to prepare 0.1 mM diluted Glucose Standard solution. Add 0,4, 8, 12, 16 and 20 μl of the 0.1 mM diluted Standard into the desired wells to generate 0, 0.4, 0.8, 1.2, 1.6 and 2.0 nmole of Standard/well respectively. Adjust the volume to 50 μl using Maltose Assay Buffer.