To determine the extracellular glucose contents secreted by the PCC 7942 derived strains, the culture broth was sampled and centrifuged at 12,000 ×g for 1 min. Then the glucose concentrations in the supernatant were calculated using the D-glucose assay kit (Megazyme) or ICS5000+ (DIONEX, Thermo Scientific, USA) ion-exchange chromatography system equipped with an electrochemical detector and a Dionex CarboPac MA1 analytical column (4×250 mm, Thermo Scientific, Waltham, MA, USA). Intracellular glucose was extracted from cellular pellets following a previous study48 (link). In brief, the cell pellets were resuspended in 1 mL 80% ethanol (volume to volume) and then incubated at 65 °C for 4 h. After centrifugation at 12,000 ×g for 5 min, the supernatant was transferred to a clean tube and dried under a stream of N2 at 55 °C. Subsequently, the dry residues were dissolved in ultrapure water, and the dissolved solution was filtered into clean vials with a 0.22 μm filtration membrane. Glucose contents in the filtrate were also calculated using the D-glucose assay kit (Megazyme) or ion chromatography.
D glucose assay kit
Manufactured by Megazyme
Sourced in Ireland, United States
The D-Glucose Assay Kit is a laboratory tool used to quantify the concentration of D-glucose in various samples. It provides a reliable and accurate method for analyzing glucose levels across a range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Amyloglucosidase, by Megazyme (3 mentions)
Glycogen colorimetric fluorometric assay kit, by Abcam (2 mentions)
Prism 9.0 software, by GraphPad (2 mentions)
Total starch assay kit, by Megazyme (2 mentions)
Bicinchoninic acid bca assay kit, by Thermo Fisher Scientific (1 mentions)
Free glycerol reagent, by Merck Group (1 mentions)
Tissuelyser 2 homogenizer, by Qiagen (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Infinity glucose hexokinase reagent, by Thermo Fisher Scientific (1 mentions)
E treh, by Megazyme (1 mentions)
Triglycerides reagent, by Thermo Fisher Scientific (1 mentions)
Sephadex g 50, by GE Healthcare (1 mentions)
Gallic acid 3 4 5 trihydroxybenzoic acid, by Merck Group (1 mentions)
α amylase from porcine pancreas type 6 b, by Merck Group (1 mentions)
Pepsin from porcine gastric mucosa, by Merck Group (1 mentions)
Amyloglucosidase from aspergillus niger, by Merck Group (1 mentions)
Power spin dx, by Unico (1 mentions)
Rice starch, by Merck Group (1 mentions)
Imark plate reader, by Bio-Rad (1 mentions)
Bca protein assay kit, by Fujifilm (1 mentions)
34 protocols using d glucose assay kit
1
Quantifying Intracellular and Extracellular Glucose
2
Hepatic Glycogen and Glucose Assay
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To determine hepatic glycogen and free glucose content, liver tissues from 24‐hour fasted young WT (n = 5) and Pygl−/− (n = 6) mice and old WT (n = 5) and Pygl−/− (n = 6) mice were homogenized. Hepatic glycogen and free glucose content were measured according to the manufacturers' instructions using a glycogen colorimetric/fluorometric assay kit (BioVision, Milpitas, CA) and a D‐glucose assay kit (Megazyme, Chicago, IL), respectively, and normalized to hepatic protein concentration measured by the bicinchoninic acid (BCA) assay kit from Thermo Fisher Scientific (Louisville, CO).
3
Quantifying Metabolite Levels in Drosophila
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Protocols for protein, lipid, and carbohydrate measurements were performed as previously described (Kwon et al., 2015 (link); Ding et al., 2021 (link)). Four female flies were used for each replicate and a minimum of three replicates were measured for each sample group. Flies were homogenized in 200 ul 1X PBS with 0.1% Triton-X and Zirconium 1 mm Oxide Beads (Next Advance Lab Products, ZROB10) using TissueLyser II homogenizer (QIAGEN). Homogenate was incubated at 70°C for 10 minutes and the supernatant was collected after centrifugation at 3,000 g for 5 min. 5 ul of supernatant was applied to Pierce™ BCA Protein Assay Kit (Thermo Scientific, 23227) for detecting protein levels. TAG and free glycerol levels were quantified from 20 ul supernatant using Triglycerides Reagent (Thermo Fisher Scientific™ - TR22421) and Free Glycerol Reagent (Sigma-Aldrich, F6428), respectively. Free glycerol was subtracted from TAG values. Glucose levels were measured from 10 ul supernatant using Infinity Glucose Hexokinase Reagent (Thermo Fisher Scientific™ - TR15421) or D-Glucose assay kit (Megazyme, K-GLUC). Trehalose levels were measured as for glucose but incubated with 0.4 ul trehalase (Megazyme, E-TREH). The amount of glucose was subtracted from trehalose read values. TAG, free glycerol, glucose, and trehalose levels were normalized to corresponding protein levels of each sample.
4
Quantifying Starch Fractions in Food
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The rapidly digestible starch (RDS) and slowly digestible starch (SDS) were measured with the method of Englyst et al. [15 ], with minor modifications as detailed by Simonato et al. [5 (link)]. The RDS and SDS contents were calculated considering the glucose released after 20 min and 120 min of incubation [15 ] by measuring the amount of glucose spectrophotometrically using a D-Glucose assay kit (GOPOD, Megazyme, Wicklow, Ireland). The resistant starch (RS) was quantified by a K-RSTAR assay kit (Megazyme, Wicklow, Ireland). The total starch content was calculated as the sum of non-resistant starch and RS following the K-RSTAR assay kit’s instructions.
5
In Vitro Glucose Hydrolysis Evaluation
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The procedure was previously described by Krawęcka et al. [24 (link)]. After in vitro digestion, the GOPOD method was used to determine the glucose content in the samples (mg glucose/g sample) (D-Glucose Assay Kit, Megazyme, USA), which was plotted as a function of time, and the areas under the hydrolysis curves (AUC) were calculated. Subsequently, the hydrolysis index (HI) was calculated as the ratio between the AUC of the sample and the AUC for the reference food (white bread). The glycemic index was predicted according to the equation:
6
Enzymatic Characterization of BcGalB
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The hydrolytic activity of BcGalB was tested on several substrates: PNP-β-glu, ONP-β-glu, PNP-α-glu, PNP-βxyl, PNP-α-man, PNP-β-man, PNP-β-fuc, PNP-α-fuc, PNP-α-rha, ONP-β-gal, PNP-β-gal, PNP-α-gal, PNP-α-ara and D-lactose. The enzyme was incubated in presence of 10 mM of each substrate under standard assay conditions. When lactose was used, the amount of free-glucose released upon hydrolysis was determined using D-Glucose Assay Kit (GOPOD Format, Megazyme) according to the manufacturer's protocol. One unit (U) is defined as the amount of enzyme required to release 1 µmol of glucose per min. In order to study the kinetic parameters of the enzyme, different concentration values of ONP-β-gal (0.1 to 20 mM) and lactose (0-500 mM) were tested. The Michaelis-Menten constant (K M ) and V max were calculated by non-linear regression analysis using GraphPad 9.0 Prism software.
7
Enzymatic Saccharification of Barley Straw
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Crude commercial enzymes and fungal enzymes were screened for their ability to enhance the saccharification yield from barley straw incubated with RME using a microassay as described previously (Badhan et al., 2014 (Badhan et al., , 2015)) . Ground barley straw was mixed in buffered suspension (composition as shown above) at a concentration of 0.5% w/v at pH 6; and 200 µl of this suspension was incubated with additive enzymes (5 mg/g substrate) and RME (5 mg/g substrate) at 39°C for 48 h. The reactions were then centrifuged at 1,500×g for 3 min; the supernatants transferred into a clean microplate and heated at 90°C for 10 min to inactivate enzymes. The amount of glucose & xylose released by the enzyme mixture was quantified using the D-Glucose Assay kit (Megazyme, Wicklow, Ireland) as described previously (Badhan et al., 2014) . The enzyme preparation that supported the greatest improvement in glucose yield of RME was selected for in-vitro batch culture validation.
8
Invertase Activity Assay in Fruits
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The invertase activity was measured according to Ohyama et al. (1995) with modifications. Whole fruits of control plants and transgenic RNAi plants at the MG stage (DAF 27-30) and the RED stage (DAF 39-45) were ground with liquid N 2 and resuspended in 20 mM Tris-HCl (pH 7.2) containing 0.5 M NaCl. After centrifugation at 12,000 x g for 20 min, the supernatants were desalted on a column of Sephadex G-50 (GE Healthcare) and used as the source of soluble enzymes. A total of 0.5 μg in 50 μl distilled deionized water was incubated with 150 μl of 3% (w/v) sucrose and 50 mM sodium acetate buffer (pH 5.2) and then boiled at 95°C for 5 min to stop the reaction. The concentration of glucose was then determined using a D-Glucose assay kit (Megazyme, Ireland).
9
Characterization of Commercial Pea Starch
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Commercial pea starch was received from Roquette (LN 30; Lestrem, France), and as noted by the manufacturer, the native pea starch contained 0.1% of crude fat and 0.5% of protein. Gallic acid (3,4,5-trihydroxybenzoic acid, 97–102%) was from Sigma-Aldrich (St. Louis, MO, USA). D-Glucose Assay Kit was purchased from Megazyme International Ltd. (Wicklow, Ireland). Pepsin from porcine gastric mucosa (371 U/mg), pancreatin from porcine pancreas (8x USP), amyloglucosidase from Aspergillus niger (319 U/mL), invertase from S. cerevisiae (334 U/mg), and α-amylase from porcine pancreas (type VI-B, 11 U/mg) were from Sigma-Aldrich (St. Louis, MO, USA). All reagents and chemicals were of analytical grade and used without further purification. Enzyme activity was defined and determined by the manufacturers, and newly purchased enzymes were used in this study.
10
Invertase Activity Assay in Yeast
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The invertase assay was performed on whole cells previously described (Silveira et al., 1996) (link). Exponentially growing cells in SMG were washed with sterile dH 2 O, transferred to shake flasks (at OD 3) with 100 mL fresh SMG or SME+0.075% glucose and incubated for 2h at 30 C and shaking at 200 rpm. Afterward the dry weight of the cultures was determined and the cells were washed in 50mM sodium acetate buffer wit 50mM NaF to block the metabolism and were then suspended till a concentration of 2.5-7.5 mg dry weight per mL. 4 mL of this cell suspension were added to a dedicated vessel thermostated at 30 C, and kept under constant aeration by flushing with air (Linde, Gas Benelux, The Netherlands) and stirring with a magnetic stirrer. The reaction was started by addition of 1 mL 1M sucrose and 1 mL reaction mix was taken at 0, 1, 2, 3 and 5 min, directly filtered using 13 mm diameter 0.22 mm pore size nylon syringe filters to remove cells and put on ice. Afterward the glucose concentration resulting from sucrose hydrolysis by invertase was determined using a D-Glucose assay kit (Megazyme, Bray, Ireland). The glucose production rate was calculated in mMol$min À1 $g dry weight À1 .
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