Glucose assay
Reagents:
- Glucose standard from Sigma, #G6918-100ml.
- PGO enzyme preparation from Sigma, #P7119-10CAP.
- Color reagent from Sigma, #F5803-50mg. Reconstitute with 20 ml H2O and use within 3 months.
- You also need a plate reader.
Glucose assay is a simple and inexpensive approach to measure glucose in laboratory. It can be applied to frozen serum samples from rodents.
1. Thaw serum at RT and spin down at 6000 RPM for 6 min.
2. Make the standard curve. In a 96-well plate, add the following amound of glucose in wells.
Glucose Concentration
0 ul for 0 mg 0.5 ul for 50 mg/dL 1 ul for 100 mg/dL 2 ul for 200 mg/dL 3 ul for 300 mg/dL 4 ul for 400 mg/dL 5 ul for 500 mg/dL 6 ul for 600 mg/dL 7 ul for 700 mg/dL 8 ul for 800 mg/dLDoublet can be used for each concentration point.
3. Add appropriate amount of H2O to fill to 25 ul for each well.
4. In the rest wells, add 24 ul H2O to each one.
5. Add 1 ul serum sample to each well.
6. Prepare 2 X PGO solution. Dissolve one capsule of PGO powder in 50 ml H2O and date it. This PGO solution is good for 1 month at 4C.
7. Based on the numder of samples, calculate the amount of PGO solution required. For each well, 250 ul PGO working solution is required. As an example, for 80 samples (including the standards), 20 ml PGO working solution is needed. For 20 ml PGO working soltuion, we need 10 ml H2O, 10 ml 2 X PGO and 320 ul color reagent solution.
8. Mix thoroughly and add to each well using multichannel pipette. Minimize the time between samples.
9. Incubate for 30 min at 37C. Alternatively, incubate at RT for 45 min.
10. Measure OD450 in a plate reader.
11. Make standard curve (usually the 600, 700 and 800 points are out of the linear range) and calculate the concentration of glucose of your samples.