Assay of relative PK activity was followed the manufacturer's protocol of Pyruvate Kinase Activity Assay Kit (Nanjingjiancheng, Nanjing, China). The lactate levels were detected through a Lactate Assay Kit (Nanjingjiancheng). Enzyme activities and lactate production were normalized on the basis of cell numbers or protein contents.
Pyruvate kinase activity assay kit
Manufactured by Nanjing Jiancheng
Sourced in China
The Pyruvate Kinase Activity Assay Kit is a laboratory instrument designed to measure the activity of the enzyme pyruvate kinase. Pyruvate kinase is an important enzyme involved in glycolysis, the metabolic pathway that converts glucose into energy. The kit provides a quantitative method to assess pyruvate kinase activity in various biological samples.
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3 protocols using pyruvate kinase activity assay kit
1
Pyruvate Kinase Activity Assay
2
Glucose Metabolism Analysis in PC Cells
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PC cells were cultured in glucose-free DMEM for 16 h, and then incubated with high-glucose DMEM under normoxic conditions for an additional 24 h. Culture medium was then removed, and intracellular glucose levels were measured using a fluorescence-based glucose assay kit (BioVision, Milpitas, California, USA) according to the manufacturer's instructions. Lactate levels were measured using a lactate oxidase-based colorimetric assay read at 540 nm according to the manufacturer's instructions (Beyotime, Wuxi, China) and normalized to cell number. Pyruvate kinase activity was measured using the Pyruvate Kinase Activity Assay Kit (Jiancheng, China).
3
Protein Expression and Enzymatic Assays
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BL21(DE3) pLYsS cells, harboring the expression vectors pET-28a-orfH79 and pET-28a, were cultured in LB medium until the optical density reached 0.6–0.8. Each culture was then divided into two subcultures. One subculture was induced with 1 mM IPTG whereas the other was used as a control. Cells were harvested at 6 h and 12 h, after induction with IPTG. Cells were disrupted by sonication and centrifuged to remove cell debris. The supernatants were used to determine the protein quantity and for use in enzymatic assays. Pyruvate kinase activity was measured using the ultraviolet chromatometry method following the protocol of the pyruvate kinase activity assay kit (Nanjing Jiancheng, China). The α-ketoglutarate dehydrogenase activity determination was performed described by Flora (Pettit et al. 1973 (link)).
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