The carboxylate ion of pyruvic acid, pyruvate, is a central molecule in various intermediary metabolic pathways. Pyruvate is produced from glucose through glycolysis, supplies energy to cells via metabolism through the tricarboxylic acid (TCA) cycle and can be converted to glucose via gluconeogenesis and lactate uptake (particularly in hepatocytes, the functional cells of the liver). It can also synthesize lipid intermediates through a key metabolic intermediate, acetyl-CoA.
BioVision's Pyruvate Assay Kit provides a simple, unique, direct and user-friendly procedure for measuring pyruvate concentration in various biological samples. In the assay, pyruvate is oxidized by pyruvate oxidase via enzymatic reactions to generate color (at 570 nm) and fluorescence (at Ex/Em of 535/587 nm). Since the color or fluorescence intensity is proportional to pyruvate content, the pyruvate concentrations can be accurately measured. The kit detects 1-1000 mM pyruvate samples.
The contents of the kit are: pyruvate assay buffer, pyruvate probe, DMSO solvent, pyruvate enzyme mix and pyruvate standard at (100 mmol/ml). Dissolve the pyruvate probe with 220 ul of DMSO before use. Dissolve pyruvate enzyme mix with 220 ul Pyruvate Assay Buffer. For the colorimetric assay, dilute the Pyruvate Standard to 1 mmol/ml by adding 10 ul of the Standard to 990 ul of Pyruvate Assay Buffer. For the fluorometric assay, dilute the standard another 10-fold to 0.1 mmol/ml by taking 10 ul of 1 mmol/ml solution into 90 ul of Pyruvate Assay Buffer. Add 0, 2, 4, 6, 8, 10 ul into a series of standards wells. Prepare test samples in 50 ul/well with Pyruvate Assay Buffer in a 96-well plate. For each well, prepare a total 50 ul Reaction Mix containing the following components: 46 ul Pyruvate Assay Buffer, 2 ul Pyruvate Probe and 2 ul Enzyme Mix. Mix well. Add 50 ul of the Reaction Mix to each well containing the Pyruvate Standard or test samples. Incubate the reaction for 30 minutes at room temperature, protect from light. Measure O.D. 570 nm for colorimetric assay (we used a Bio-Rad 680 plate reader) or fluorescence at Ex/Em of 535/590 nm in a microplate reader. Plot standard curve nmol/well vs. O.D. 570 nm readings. Then apply the sample readings to the standard curve to get pyruvate amount in the sample wells.
We have used the pyruvate assay kit in our laboratory for identifying the concentration of the molecule in hepatocyte culture supernatants. The ease of detection, quantification and comparison of positive experimental samples to negative controls (i.e. basal media without exposure to cells) has been excellent in our experience. We truly recommend this assay kit for measurement of pyruvate in cell culture media samples.