The glucose level was determined using a glucose-oxidase assay kit (Sigma). The triglyceride and cholesterol levels were determined according to the kit instruction.
Glucose oxidase assay kit
Manufactured by Merck Group
Sourced in United States
The Glucose oxidase assay kit is a laboratory equipment used to measure the concentration of glucose in a sample. It utilizes the enzymatic reaction between glucose and glucose oxidase to quantify the amount of glucose present. The kit provides the necessary reagents and protocols to perform this analysis.
Automatically generated - may contain errors
Lab products found in correlation
Sigma protease inhibitor, by Merck Group (1 mentions)
Diprotinin a, by MP Biomedicals (1 mentions)
Prism 5, by GraphPad (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
M199 medium, by Corning (1 mentions)
Porcine trehalase, by Merck Group (1 mentions)
Lactate assay kit, by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Hexane, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
N butanol, by Merck Group (1 mentions)
Acarbose, by Merck Group (1 mentions)
Ch2cl2, by Merck Group (1 mentions)
Spectronic 300 array spectrophotometer, by PerkinElmer (1 mentions)
Ft ir 1760x spectrophotometer, by PerkinElmer (1 mentions)
Microtof mass spectrometer, by Bruker (1 mentions)
Etoac, by Merck Group (1 mentions)
Yeast α glucosidase, by Merck Group (1 mentions)
3 4 5 dimethyl thiazol 2 yl 5 diphenyl tetrazolium bromide mtt, by Merck Group (1 mentions)
10 protocols using glucose oxidase assay kit
1
Biochemical Metabolite Measurement
2
Biomarker Sampling and Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At each time point, approximately 5 mL of venous blood was collected. Samples were aliquoted into a 2 mL into a K2EDTA plasma vacutainer, 2 mL into a serum vacutainer, and 1 mL into an Eppendorf tube containing EDTA, diprotinin-A (0.034 g/L blood; MP Biomedicals), sigma protease inhibitor (1 g/L blood; Sigma Aldrich), and Roche Pefabloc® SC (1 g/L of blood, Sigma Aldrich) to preserve analytes. Tubes were centrifuged for 15 min at 4°C. Supernatant was removed from each sample and transferred into a new labeled Eppendorf tube for storage at −80°C until analysis. Blood samples collected in Study A were assessed by a custom Human Metabolic Array [C-peptide, glucagon, gastric inhibitory peptide (GIP), glucagon-like peptide-1 (GLP-1 active), insulin and peptide YY (total)] (Milliplex Multiplex, Eve Technologies, University of Calgary). A glucose oxidase assay kit used plasma samples to measure plasma glucose concentrations at all time points (Sigma Aldrich). Serum paracetamol concentrations, determined using a paracetamol assay kit (Cambridge Life Science), were used to track gastric emptying. Several studies have demonstrated paracetamol concentrations to be a valid measure of gastric emptying (10 (link), 11 (link)).
3
Quantifying Glucose and Glycogen in Flies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Seven flies were homogenized in ice-cold PBS and glucose and glycogen contents (N = 4–5) were measured using a Glucose Oxidase Assay kit (Sigma-Aldrich®), according to the manufacturer’s protocol at 540 nm. Glycogen was determined by measuring glycogen + glucose content in presence of amyloglucosidase and subtracting the glucose concentration (samples without amyloglucosidase) as described elsewhere46 (link). Glucose and glycogen contents are expressed as means of μg of glucose or glycogen per mg of proteins ± s.e.m.
4
Kinetic Analysis of MhGgH Enzyme
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The effect of temperature and pH on the catalytic activity of MhGgH was evaluated by quantifying the glucose released upon hydrolysis of GG using the Glucose Oxidase Assay Kit (Sigma–Aldrich) as described previously (Alarico et al., 2014 ▸ ). The temperature profile of MhGgH was traced from 20 to 60°C using the standard reaction conditions (see Section 2.7). The effect of pH on the activity of MhGgH was determined at 55°C using 20 mM sodium acetate (pH 4.0–5.5) or 20 mM sodium phosphate (pH 5.8–7.0) buffer.
Kinetic parameters for the MhGgH-catalysed hydrolysis of GG and MG were determined by quantifying the release of glucose (as described above) or mannose (using the K-MANGL 01/05 assay kit; Megazyme), respectively. A constant enzyme concentration (2.75 µM) was incubated with increasing concentrations of GG (0–35 mM) or MG (0–150 mM) in 20 mM sodium phosphate pH 6.0, 100 mM KCl, 5 mM MgCl2 at 50°C. The maximum velocity (Vmax) and half constant (K0.5) were calculated with Prism 5.0 (GraphPad Software) using the allosteric sigmoidal equation. Experiments using GG were performed in triplicate, while those using MG as substrate were performed in duplicate.
Kinetic parameters for the MhGgH-catalysed hydrolysis of GG and MG were determined by quantifying the release of glucose (as described above) or mannose (using the K-MANGL 01/05 assay kit; Megazyme), respectively. A constant enzyme concentration (2.75 µM) was incubated with increasing concentrations of GG (0–35 mM) or MG (0–150 mM) in 20 mM sodium phosphate pH 6.0, 100 mM KCl, 5 mM MgCl2 at 50°C. The maximum velocity (Vmax) and half constant (K0.5) were calculated with Prism 5.0 (GraphPad Software) using the allosteric sigmoidal equation. Experiments using GG were performed in triplicate, while those using MG as substrate were performed in duplicate.
5
Evaluating Enzyme Activities in Biological Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The activities of versatile peroxidases (VPs; Mn-dependent and Mn-independent activity) and superoxide dismutase (SOD) were evaluated using a microplate assay according to Rybczyńska-Tkaczyk et al. (2020) [23 (link)]. The activities of catalase (CAT) and glucose oxidase (GOX) were estimated using a catalase assay kit (Merc Millipore, Burlington, MA, USA) and a glucose oxidase assay kit (Sigma Aldrich), respectively. The GOX assay is based on D-glucose oxidation and production of H2O2, which reacts with the probe, generating a colorimetric (A570 nm) product proportional to the GOX activity. One unit of GOX is defined as the amount of the enzyme that generates 1 µmole of H2O2 per minute at 37 °C. The protein concentration was determined according to the Bradford method [63 (link)] using a protein assay kit (BioRad, Hercules, CA, USA).
6
Isolation and Culture of Mouse Hepatocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary hepatocytes were isolated from the livers of male 8-week-old mice using a portal vein collagenase (Sigma‒Aldrich) perfusion method as described previously19 (link). Primary mouse hepatocytes were maintained in M199 medium (Cellgro, Herdon, VA, USA). The cells were utilized for adenoviral infection and FSK (10 μM) or melatonin (500 μM) treatment. Glucose production was performed using a glucose oxidase assay kit (Sigma‒Aldrich) as mentioned previously19 (link).
7
Hemolymph Extraction and Glucose/Trehalose Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For hemolymph extractions, 30-40 female flies were perforated with a 27G needle and placed in a 0.5 mL Eppendorf tube perforated with a 27G needle. The Eppendorf tubes were placed inside 1.5 mL Eppendorf tubes and centrifuged for 5 min at 5,000 × g at 4°C to collect hemolymph. A 1-μL aliquot of the collected hemolymph was diluted in 99 µl of trehalase buffer (5 mM Tris pH 6.6, 137 mM NaCl, 2.7 mM KCl), followed by heat treatment for 5 min at 70°C.
A 30-μL portion of supernatant was used to measure circulating glucose levels with glucose oxidase assay kit (Sigma-Aldrich, GAGO-20) according to the manufacturer's instructions, as previously described 101 . Trehalose measurement was performed by diluting 30 μL of supernatant with 30 μL of trehalase buffer and 0.09 uL of porcine trehalase (Sigma-Aldrich, T8778-1UN). The solution was then incubated overnight in 37 °C. A 30 µL aliquot of each sample was used to measure circulating trehalose levels with the glucose oxidase assay kit.
A 30-μL portion of supernatant was used to measure circulating glucose levels with glucose oxidase assay kit (Sigma-Aldrich, GAGO-20) according to the manufacturer's instructions, as previously described 101 . Trehalose measurement was performed by diluting 30 μL of supernatant with 30 μL of trehalase buffer and 0.09 uL of porcine trehalase (Sigma-Aldrich, T8778-1UN). The solution was then incubated overnight in 37 °C. A 30 µL aliquot of each sample was used to measure circulating trehalose levels with the glucose oxidase assay kit.
8
Quantification of Metabolites in Cell Supernatants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Metabolites were quantified from cell supernatants using the Glucose Oxidase (GO) Assay Kit and the Lactate Assay Kit from Millipore Sigma-Aldrich (Saint-Quentin Fallavier, France). Assays were performed according to the manufacturer’s instructions and results were normalized to protein concentration per sample (DC Protein Assay; Bio-Rad, Marnes-la-Coquette, France).
9
Spectroscopic Characterization of Novel Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
UV spectra were measured on a Milton Roy Spectronic 300 Array spectrophotometer, and the IR was recorded on a Perkin-Elmer FT-IR 1760x spectrophotometer (Boston, MA, USA). High-resolution electrospray ionization mass spectra (HR-ESI-MS) were recorded with a Bruker micro TOF mass spectrometer (Billerica, MA, USA). NMR spectra were obtained with a Bruker Avance DPX-300 or Avance III HD 500FT-NMR spectrometer (Billerica, MA, USA). MeOH, EtOAc, n-butanol, hexane, CH2Cl2, yeast α-glucosidase, p-NPG, glucose oxidase (GO) assay kit, sodium dodecyl sulfate (SDS), 3-(4,5- dimethyl thiazol-2-yl)-5-diphenyl tetrazolium bromide (MTT), and acarbose were obtained from Sigma-Aldrich (St. Louis, MO, USA). Alpha minimal essentialmedium (α-MEM), fetal bovine serum (FBS), and penicillin-streptomycin (10,000 IU/mL) were purchased from Thermo Fisher Scientific (Grand Island, NY, USA). Insulin (100 IU/mL) was acquired from Biocon (Bangalore, India). Silica gel (SiO2) and TLC plates were obtained from Merck (Darmstadt, Germany), and Sephadex LH-20 was from Pharmacia (Piscataway, NJ, USA).
10
Quantifying Intracellular Glycogen and Trehalose
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Glycogen and trehalose content were determined by a modified method developed by Masuko et al.44 (link). 5 mg of isolated cells in 50 µl H2O was mixed with 200 µl 313 mM sodium carbonate and incubated at 90 °C for 4 h. Then 600 µl 0.2 M sodium acetate and 150 µl of 1 M acetic acid were added, and the sample was vortexed and centrifuged (2 min, 5000g). 200 µl of the supernatant was mixed with either 5 µl 2 U/ml trehalose (for trehalose assay; Megazyme) or 5 µl 50 U/ml amyloglucosidase (for glycogen assay; Sigma) and incubated overnight at 37 °C or 52 °C, respectively. Glucose released by enzymatic degradation was quantified using the glucose oxidase (GO) assay kit (Sigma).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!