Pyruvate kinase (PK) regulates cell metabolism and catalyzes the last reaction in glycolysis converting phosphoenolpyruvate (PEP) to pyruvate. Mutations in PK causes pyruvate kinase deficiency which can lead to hemolytic anemia, pulmonary hypertension, and various cancers. Monitoring PK activity is important in diagnostics and management of PK deficiency.

Pyruvate Kinase Activity Assay Kit provides a simple, reproducible, and sensitive tool of measuring pyruvate kinase activity in plasma, serum, cell lysates, urine, saliva, and other biological liquid samples. This assay is based on a coupled enzyme reaction. First PEP and ADP are catalyzed by pyruvate kinase to generate pyruvate and ATP. The generated pyruvate is then oxidized to produce colored product which can be quantified using a spectrophotometer or a microtiter plate reader at 570 nm. The change in absorbance is directly proportional to Pyruvate Kinase activity in the sample.

• Microplate reader capable of measuring absorbance at 570 nm
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Tubes to prepare sample dilutions
• 5 mL tubes
• MilliQ water or another type of double distilled water (ddH₂O)
• Microplate sealer or plastic wrap

1. Add 50 µL of the diluted pyruvate standards, samples, or positive control to the 96-well microtiter plate.
2. Add 50 µL Reaction Mix to each standard and one half of the paired sample wells, mix thoroughly.
3. Add 50 µL of Control Mix to other half of the paired sample wells. Carefully shake the plate for a few seconds to mix.
4. Kinetic reading: Immediately start measuring absorbance at 570 nm continuously at 25°C and record data every 4 minutes for 32 minutes.