Amplite® Colorimetric Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Assay Kit

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

1. Prepare the test samples, GAPDH Positive Control, and the serially diluted NADH standards (50 μL).

2. Add the GAPDH working solution (50 μL).

3. Incubate at room temperature for 10-30 minutes.

4. Measure the absorbance at 450 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. Add 200 µL of PBS buffer to the vial of NADH standard (Component D) to make a 2 mM (2 nmol/µL) NADH stock solution. Store the solution at -80°C. Avoid repeated freeze/thaw cycles.

1. To prepare a GAPDH stock solution, reconstitute the GAPDH Positive Control (Component E) by adding 40 µL of ddH2O. Mix well by pipetting and store at –20 °C.

   Note: Must be used within 2 months of reconstitution.

1. To prepare a 50X GAPDH Substrate stock solution, add 100 µL of ddH2O to the vial containing the GAPDH Substrate (Component C). After mixing, store the solution at -20°C. Avoid repeated freezing and thawing.

1. Add 1 mL of the GAPDH Probe (Component B) to the bottle containing 4 mL of the GAPDH Assay Buffer (Component A), and mix well.

2. Add 50 µL of the GAPDH Substrate stock solution to the same bottle and mix well.

   Note: This GAPDH working solution should be freshly prepared before each experiment and protected from light. A 5 mL solution is enough for 100 tests. Please prepare the necessary amount of GAPDH working solution based on this proportion.

1. Prepare one or more GAPDH positive control samples along with the test sample. The recommended concentration for the GAPDH positive control is 50 mU/mL in PBS + 0.1% BSA.

Table 1. Layout of GAPDH standards and test samples in a 96-well clear bottom microplate. (STD = NADH Standards (STD1-STD7, 3.125 to 200 uM), BL= Blank Control, TS = Test Samples.)

Table 2. Reagent composition for each well.

1. Prepare the NADH standards (STD1-7), blank controls (BL), GAPDH Positive Control, and test samples (TS) according to the layout provided in Tables 1 and 2. When using a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

2. Add 50 µL of GAPDH Working Solution to each well containing the NADH standard, blank control, GAPDH Positive Control, and test samples. For a 384-well plate, add 25 µL of GAPDH Working Solution to each well instead.

3. Incubate at room temperature for 10–30 minutes, protected from light.

4. Monitor the absorbance intensity with an absorbance microplate reader at 450 nm.