Enzymatic analysis of plasma triglyceride (TG) and total cholesterol (TC) was performed by using commercial kits (Asan Pharm Co., Seoul, Korea). Free fatty acid (FFA) was also measured by using an enzymatic assay (ABcam, Cambridge, MA, USA). After extracting hepatic and muscle lipids 22, the dried lipid residues were dissolved in 1 mL of ethanol for triglyceride, TC, and FFA measurements. For emulsification, Triton X-100 and sodium cholate solution were added to 200 µL of dissolved lipid solution. Triglyceride, TC, and FFA were measured by using the same kit as that used for plasma lipid analysis.
Enzymatic assay
Manufactured by Abcam
Sourced in United States
Enzymatic assay is a laboratory technique used to measure the activity or concentration of a specific enzyme in a sample. It involves the use of a substrate that is converted by the enzyme into a measurable product, which can be detected and quantified. This process provides a direct way to assess the function and abundance of enzymes in various biological samples.
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Lab products found in correlation
6 protocols using enzymatic assay
1
Quantifying Plasma and Tissue Lipid Profiles
2
Triglyceride Quantification Protocol
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For total TG quantification, lipids were extracted according to the manufacturer's instructions. Briefly, MPBAs were homogenized in 1 mL of solution containing 5% NP-40 substitute (USB Corporation, Cleveland, OH, USA). The homogenates were then slowly heated to 80°C–100°C in a heating block for 2–5 min until the NP-40 became cloudy, followed by cooling to room temperature. Samples were subsequently centrifuged for 3 min to remove the insoluble materials. TG levels were measured by enzymatic assay (Abcam) and normalized to their respective protein concentrations.
3
Colorimetric Assays for Lactate and Glucose
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Lactate concentrations were analyzed using a colorimetric enzymatic assay (Sigma-Aldrich). The lactate determination kit comes with premade lactate assay buffer, lactate probe, enzyme mix, and standards. Samples were prepared and analyzed as per the manufacturer's protocol. Briefly, CM samples were diluted 1:50 in lactate assay buffer. Sample (50 mL) was incubated with 50 mL reaction mixture for 30 minutes at RT. Absorbance was measured at 570 nm. Similarly, glucose concentrations were measured using an enzymatic assay as per the manufacturer's instructions (Abcam). Conditioned media were diluted 1:50 in glucose assay buffer and incubated with an equal volume of reaction buffer, at 37 C for 30 minutes. Absorbance was measured at 570 nm.
4
Serum Creatinine and Urine 8-epi-PGF2α Measurement
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Creatinine concentration in serum was determined using an enzymatic assay (Biovision, Milpitas, California, US), according to the manufacturers’ instructions.
Urine was collected in metabolic cages before death and 8-epiprostaglandin-F2α (8-epi-PGF2α) was calculated using an EIA kit (8-isoprostane; Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturers’ instructions.
Urine was collected in metabolic cages before death and 8-epiprostaglandin-F2α (8-epi-PGF2α) was calculated using an EIA kit (8-isoprostane; Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturers’ instructions.
5
Enzymatic Assay for L-2HG Detection
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The enzymatic assay was obtained from BioVision. For cell-independent experiments, α-KGDH enzyme and necessary co-enzymes were incubated with increasing concentrations of L-2HG. L-2HG was synthesized as previously described (Shim et al., 2014 (link)). Optical density readings at 450 nm were taken every 5 min over 70 min.
6
Measurement of ALP Activity in MC3T3-E1 Cells
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ALP activity was measured by enzymatic assay (Biovision, Minneapolis, MN, USA). Briefly, MC3T3-E1 cells were seeded on 24-well plates, treated with various concentrations of genistein (5, 10, or 20 µM) or transfected with siRNAs. After 3 days, the cells were washed with phosphate-buffered saline (PBS) and lysed in buffer. The cell lysates were centrifuged, and the supernatants were subjected to the assay. Absorbance of p-nitrophenol at 520 nm was determined on a microplate spectrophotometer.
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