The Hexokinase Colorimetric Assay Kit (Sigma-Aldrich) was used according to manufacturer's instructions. Briefly, DFCs were washed with PBS, scraped in assay buffer from the kit, and centrifuged. The supernatant was further used to determine hexokinase activity in a colorimetric reaction, which was measured spectrophotometrically at OD = 450 nm in kinetic mode.
Hexokinase colorimetric assay kit
Manufactured by Merck Group
Sourced in United States
The Hexokinase Colorimetric Assay Kit is a laboratory equipment product that measures the activity of the enzyme hexokinase. Hexokinase is an important enzyme involved in glucose metabolism. The kit uses a colorimetric method to quantify hexokinase activity in samples.
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Lab products found in correlation
Lactate dehydrogenase activity assay kit, by Merck Group (2 mentions)
Glucose uptake glo assay kit, by Promega (1 mentions)
Nad nadh assay kit wst, by Dojindo Laboratories (1 mentions)
Spectramax m5 plate reader, by Molecular Devices (1 mentions)
Enzymatic assay of pyruvate kinase kit, by Merck Group (1 mentions)
Gdh kit, by Abcam (1 mentions)
Phosphofructokinase activity colorimetric assay kit, by Merck Group (1 mentions)
Glycerol assay kit, by Merck Group (1 mentions)
Free fatty acid assay kit, by Cell Biolabs (1 mentions)
Serum triglyceride determination kit, by Merck Group (1 mentions)
Lactate assay kit, by Abcam (1 mentions)
Pyruvate kinase activity assay kit, by Merck Group (1 mentions)
Phosphofructokinase pfk activity colorimetric assay kit, by Merck Group (1 mentions)
Gapdh activity assay kit, by Merck Group (1 mentions)
Bca 1 kit, by Merck Group (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Pyruvate dehydrogenase activity assay kit, by Merck Group (1 mentions)
Thenoyltrifluoroacetone, by Merck Group (1 mentions)
Atp determination kit, by Thermo Fisher Scientific (1 mentions)
Jc 1 dye, by Thermo Fisher Scientific (1 mentions)
16 protocols using hexokinase colorimetric assay kit
1
Hexokinase Activity in Differentiated Cells
2
Brain Tissue Hexokinase Activity Assay
3
Metabolic Analysis of IMS32 Cells
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IMS32 cells were seeded at a density of 9 × 103 cells/cm2 and maintained for 30 or 60 min under each condition described as Measurement of ROS production. GAPDH activity, hexokinase activity, glucose uptake, and NAD and NADH levels under each culture condition were quantified using Glyceraldehyde 3 Phosphate Dehydrogenase assay kit (AbCam), Hexokinase Colorimetric Assay Kit (Sigma-Aldrich), Glucose Uptake-Glo Assay kit (Promega) and NAD/NADH Assay Kit-WST (DOJINDO Laboratories, Kumamoto, Japan), respectively, according to the manufacturer’s instructions.
4
Hexokinase Activity Quantification
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Hexokinase activity was measured using the hexokinase colorimetric assay kit (Sigma-Aldrich. St. Louis, MO) according to the manufacturer’s instructions. Absorbance was measured at 563 nm using a SpectraMax M5 plate reader (Molecular Devices). One unit of hexokinase is the amount of enzyme that will generate 1.0 mM of NADH per min at pH 8.0 and room temperature. The results were normalized to the amount of total protein compared to the sham.
5
Measurement of Glucose Uptake and Enzyme Activities
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The uptake of glucose was measured via radiolabeling whole cells with 1 µCi-deoxy-D-[3H]-glucose (PerkinElmer, Waltham, MA, USA) [76 (link)]. The results are expressed as picomoles of 2-deoxy-D-[3H]-glucose/mg cell proteins. The HK activity was measured with the Hexokinase Colorimetric Assay Kit (Sigma-Aldrich). The activities of the phosphofructokinease-1 (PFK1) assay and enolase were measured spectrophotometrically as reported in [77 (link)]. The activity of PK was detected with the Enzymatic Assay of Pyruvate Kinase kit (Sigma-Aldrich). The results are expressed as nmoles NADH/min/mg cell proteins (HK) or nmoles NAD+/min/mg cell proteins (PFK1, enolase, PK).
6
Enzymatic Assays for Glycolysis and Mitochondrial Metabolism
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Activity of hexokinase, the rate-limiting enzyme in the glycolytic pathway, was measured using a hexokinase colorimetric assay kit (Sigma-Aldrich, St. Louis, MO, USA) as per the vendor’s protocol. Briefly, it is a coupled enzyme assay, resulting in the production of NADH, which reduces a colorless probe, producing a colorimetric (450 nm) product proportional to the hexokinase activity.
Activity of glutamate dehydrogenase (GDH), a mitochondrial enzyme, was measured using a GDH kit (Abcam, Cambridge, UK) as per the manufacturer’s instructions. Briefly, mitochondria from the different tissues were treated with a reaction buffer, containing glutamate as a specific substrate and the NADH generated was determined spectrophotometrically using the UV-1800 spectrophotometer (Schimadzu Corporation, Kyoto, Japan) at 450 nm.
Activity of glutamate dehydrogenase (GDH), a mitochondrial enzyme, was measured using a GDH kit (Abcam, Cambridge, UK) as per the manufacturer’s instructions. Briefly, mitochondria from the different tissues were treated with a reaction buffer, containing glutamate as a specific substrate and the NADH generated was determined spectrophotometrically using the UV-1800 spectrophotometer (Schimadzu Corporation, Kyoto, Japan) at 450 nm.
7
Enzymatic Profiling of Tea Leaves
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The total protein was extracted from tea leaves with the M5 Plant Protein Extraction Kit (Mei5 Biotechnology, Co., Ltd, Beijing, China). The activities of enzymes, including SPP, SPS, neutral INV, SUS, CHI, F3H, ANR, F3′H, UFGT, and F3′5′H, were measured using the corresponding ELISA kits (Shanghai SUER Biological Technology, Co., Ltd, Shanghai, China). The activity of HXK was measured with a hexokinase colorimetric assay kit (Sigma–Aldrich, St Louis, MO, USA).
8
Enzymatic Activity Assays for Glycolysis
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PFK activity was assessed through the Phosphofructokinase Activity Colorimetric Assay Kit (Sigma, St Louis, MO, USA) according to the manufacturer's instructions. In the preexperiment, 30 min was proved to be the most suitable. HK2 activity was assessed through the Hexokinase Colorimetric Assay Kit (Sigma).
9
Metabolic Profiling of Amniotic Fluid and Plasma
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Biochemical metabolic parameters in amniotic fluid and plasma were measured using
commercially available kits according to the manufacturer’s protocols;
for glucose by the hexokinase colorimetric assay kit (Sigma-Aldrich, Saint
Louis, MO, USA), for glycerol by the glycerol assay kit (Sigma-Aldrich, Saint
Louis, MO, USA), for triglyceride by the serum triglyceride determination kit
(Sigma-Aldrich, Saint Louis, MO, USA), for lactate by the lactate assay kit
(BioVision, Mountain View, CA, USA) and for FFA by the free fatty acid assay kit
(Cell Biolabs, San Diego, CA, USA). Osmolality of serum and amniotic fluid was
determined by freezing point method with Multi Osomometer (Precision System
Inc., Natick, MA, USA). Electrolyte (Na, K and Cl) concentrations in amniotic
fluid were determined by ion selective electrode method using Toshiba TBA 200FR
(Toshiba Medical Systems Co., Ltd., Tokyo, Japan). Other chemical assays of
amniotic fluids were done using automation system of diagnostic and laboratory
medicine in Dong-A university.
commercially available kits according to the manufacturer’s protocols;
for glucose by the hexokinase colorimetric assay kit (Sigma-Aldrich, Saint
Louis, MO, USA), for glycerol by the glycerol assay kit (Sigma-Aldrich, Saint
Louis, MO, USA), for triglyceride by the serum triglyceride determination kit
(Sigma-Aldrich, Saint Louis, MO, USA), for lactate by the lactate assay kit
(BioVision, Mountain View, CA, USA) and for FFA by the free fatty acid assay kit
(Cell Biolabs, San Diego, CA, USA). Osmolality of serum and amniotic fluid was
determined by freezing point method with Multi Osomometer (Precision System
Inc., Natick, MA, USA). Electrolyte (Na, K and Cl) concentrations in amniotic
fluid were determined by ion selective electrode method using Toshiba TBA 200FR
(Toshiba Medical Systems Co., Ltd., Tokyo, Japan). Other chemical assays of
amniotic fluids were done using automation system of diagnostic and laboratory
medicine in Dong-A university.
10
Glycolysis Enzyme Activities in A375 Cells
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