1. Standard Curve Preparations: For the colorimetric assay: Dilute the Pyruvate Standard to 1 nM/µL by adding 10 µL of the Standard to 990 µL of Assay Buffer, mix well. For the fluorometric assay: Dilute the Pyruvate Standard to 1 nM/µL as for the colorimetric assay. Then dilute the standard another 10-fold to 0.1 nM/µL by taking 10 µL into 90 µL of Pyruvate Assay Buffer. Mix well. Add 0, 2, 4, 6, 8, 10 µL of the diluted standard into a series of wells. Adjust volume to 50 µL/well with Assay Buffer to generate 0, 2, 4, 6, 8, and 10 nM/well of the Pyruvate Standard for the colorimetric assay, or 0, 0.2, 0.4, 0.6, 0.8, and 1.0 nM/well for the fluorometric assay.
2. Sample and Positive Control Preparations: Serum can be directly added into sample wells. Tissues or cells can be extracted with 4 volume of the Assay Buffer, centrifuge to get clear extract. Add samples directly into 96 well plate, bring volume to 50 µL/well with PK Assay Buffer. We suggest testing several doses of your sample to ensure the readings are within the linear range. For the positive control (optional), add 5 µL positive control solution to wells, adjust volume to 50 µL/well with Assay Buffer.
3. Reaction Mix Preparation: Mix enough reagents for the number of standard and assays to be performed. For each well, prepare a total 50 µL Reaction Mix containing: Pyruvate Kinase Measurement Background Control* Assay Buffer 44 µL 46 µL Substrate Mix 4 µL ----------- Enzyme Mix 4 µL 2 µL OxiRed™ Probe** 4 µL 2 µL *Pyruvate in the sample will generate background. If significant amount of pyruvate is in your sample, the background control should be performed. The background readings are then subtracted from your sample readings. ** For fluorescent assay, dilute the probe 10X to reduce fluorescence background.
4. Add 50 µL of the reaction mix to each well containing the pyruvate standard, samples and controls, mix well.
5. Measure O.D. 570 nm or fluorescence Ex/Em = 535/587 nm at T 1 to read A 1, measure again at T 2 after incubating the reaction at 25 °C for 10-20 min (or incubate longer time if the PK activity is low in sample) to read A 2, protect from light. The signal increase is due to pyruvate generated by PK, deltaA = A 2 - A 1 Note: It is essential to read A 1 and A 2 in the reaction linear range. It will be more accurate if you read the reaction kinetics. Then choose A 1 and A 2 in the reaction linear range.
6. Calculation: Subtract 0 standard readings from the standards. Plot the pyruvate standard curve. Apply the deltaA to the standard curve to get B nmol of pyruvate generated between T 1 and T 2 by PK in the reaction wells. PK calculation: B PK Activity = x Sample Dilution Factor = nM/min/mL = mU/mL (T1-T2) x V Where: B is the pyruvate amount from pyruvate standard curve (in nmol). T 1 is the time of the first reading (A1) (in min). T 2 is the time of the second reading (A2) (in min). V is the sample volume added into the reaction well (in ml). Unit definition: One unit pyruvate Kinase is the amount of enzyme transfer of phosphate group from PEP to ADP, yielding 1.0 µM of pyruvate per minute at 25 °C.