Pyruvate Assay for Estimation of Pyruvate Levels in Cellular Fractions of Worms
Published: April 30, 2023
Abstract
Source: Yanase, S. et al. Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans. J. Vis. Exp. (2018).
This video describes a colorimetric assay for quantitatively measuring pyruvate in the cellular fractions of Caenorhabditis elegans. Utilizing the hydrogen peroxide produced from the enzymatic reaction of pyruvate oxidase with cellular pyruvate, a chromogenic dye is oxidized to produce a colored product, measured using colorimetry to determine the pyruvate concentration.
Protocol
1. Synchronized Culture of C. elegans Before seeding, culture the Escherichia coli (E. coli) strain OP50 overnight at 37 °C in 300 mL of Luria-Bertani (LB) broth liquid medium. Store the cultured OP50 at -4 °C. To make LB broth liquid medium, use 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 1.5 mL of 1 N NaOH, and add to 1 L with deionized water. Autoclave. NOTE: OP50 and C. elegans strains are available from the C…
Representative Results
Figure 1. Process for the extraction of cellular metabolites. (A) 1,500-3,000 worms were placed on an NGM agar plate (90-mm Petri dish). Extraction from worms on five plates was sufficient for the colorimetric assays. (B) Worms collected from the five plates of 3,000 and 1,500 worms per plate are indicated on the left and right side of panel, respectively. Both t…
Offenlegungen
The authors have nothing to disclose.
Materials
| EnzyChrom Pyruvate Assay Kit | BioAssay Systems | #EPYR-100 | colorimetric/ fluorometric 100 assays; Store at -20°C |
| Trichloroacetic Acid | Wako Pure Chemical | #207-04955 | store at room temperature |
| Teflon homogenizer | Iwaki/Pyrex | #358034 (Wheaton) | Instead of Iwaki/Pyrex, available by Wheaton |
Tags
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Pyruvate Assay for Estimation of Pyruvate Levels in Cellular Fractions of Worms. J. Vis. Exp. (Pending Publication), e21320, doi: (2023).