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Trehalose assay kit

Manufactured by Megazyme
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Sourced in Ireland, United States

The Trehalose Assay Kit is a quantitative enzymatic-based kit designed to measure the concentration of trehalose in various sample types. The kit provides the necessary reagents and protocols to accurately determine trehalose levels.

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Lab products found in correlation

Du 800 uv vis spectrophotometer, by Beckman Coulter (1 mentions) Amyloglucosidase, by Merck Group (1 mentions) Amyloglucosidase, by Megazyme (1 mentions) Thiazolyl blue tetrazolium bromide, by Merck Group (1 mentions) Phosphate buffered saline tablet, by Merck Group (1 mentions) Carbonyl cyanide m chlorophenylhydrazone cccp, by Merck Group (1 mentions) Epidermal growth factor (egf), by Merck Group (1 mentions) O phenylenediamine dihydrochloride, by Merck Group (1 mentions) Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Trypsin edta, by Thermo Fisher Scientific (1 mentions) Sucrose, by Merck Group (1 mentions) Glucagon, by Merck Group (1 mentions) Hydrocortisone, by Merck Group (1 mentions) Fibronectin, by BD (1 mentions) Bca assay kit, by Thermo Fisher Scientific (1 mentions) Infinite m200 pro, by Tecan (1 mentions) Elx808 microplate reader, by Agilent Technologies (1 mentions) Sodium tripolyphosphate, by Merck Group (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions)

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Trehalose Kit assays

23 protocols using trehalose assay kit

1

Quantifying Intracellular Trehalose in S. salinus

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Trehalose was detected via a coupled enzyme reaction assay using the “Trehalose Assay Kit” purchased from Megazyme (Wicklow, Ireland). In this assay, the trehalose concentration of the samples is quantified by the photometrical determination of NADPH at a wavelength of 340 nm after the trehalase is stoichiometrically converted into gluconate-6-phosphate and NADPH by the activity of the enzymes trehalose, hexokinase, and glucose-6-phosphate dehydrogenase. To avoid background reactions of contaminating sugars, an alkaline borohydride reduction of our samples was performed. The alkaline borohydride reduction and the enzymatic reaction were performed as detailed in the manual of the Megazyme Trehalose Assay Kit with the exception that we calculated the trehalose concentrations of the samples using a standard curve derived from the parallel measurement of trehalose standards with concentrations ranging between 0 and 1000 μM. The individual values are given as intracellular trehalose concentration (μM) in a S. salinus M19-40 culture that corresponds to an OD600 value of 1.
León M.J., Hoffmann T., Sánchez-Porro C., Heider J., Ventosa A, & Bremer E. (2018). Compatible Solute Synthesis and Import by the Moderate Halophile Spiribacter salinus: Physiology and Genomics. Frontiers in Microbiology, 9, 108.
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2

Trehalose Concentration Assay

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Strains were cultured overnight in 10 ml LB. Bacteria were centrifuged, washed, and resuspended in 500 µl double-distilled water (ddH2O). The lysate was prepared by treating the bacteria for 30 min at 95°C. The trehalose assay kit (Megazyme) was used to measure intracellular trehalose concentrations in the supernatant as described by the manufacturer.
Felgner S., Frahm M., Kocijancic D., Rohde M., Eckweiler D., Bielecka A., Bueno E., Cava F., Abraham W.R., Curtiss R I.I.I., Häussler S., Erhardt M, & Weiss S. (2016). aroA-Deficient Salmonella enterica Serovar Typhimurium Is More Than a Metabolically Attenuated Mutant. mBio, 7(5), e01220-16.
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3

Trehalose Quantification in Nanoparticles

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To determine EE (the weight percentage of trehalose in nanoparticles out of trehalose/PI initially fed for encapsulation) and LC (the weight percentage of trehalose in nanoparticles out of the total weight of both trehalose and nanoparticles), the amount of trehalose concentration in various samples was determined using a trehalose assay kit (Megazyme, Wicklow, Ireland) by following the manufacturer’s instructions. In brief, trehalose in a sample was phosphorylated using hexokinase and adenosine-5’-triphosphate (ATP) into glucose-6-phosphate (G-6-P). In the presence of glucose-6-phosphate dehydrogenase (G6P-DH), the produced G-6-P was oxidized by nicotinamide-adenine dinucleotide phosphate (NADP+) into gluconate-6-phosphate and reduced nicotinamide-adenine dinucleotide phosphate (NADPH). The absorbance at 340 nm of NADPH was then measured using a Beckman Coulter DU800 UV-Vis spectrophotometer to determine the amount of trehalose in the sample. The amount of PI was quantified colorimetrically by dispersing nPI in DI water and measure the absorbance at 494 nm with a Beckman Coulter DU800 UV-Vis spectrophotometer (Indianapolis, IN, USA).
Rao W., Huang H., Wang H., Zhao S., Dumbleton J., Zhao G, & He X. (2015). Nanoparticle-Mediated Intracellular Delivery Enables Cryopreservation of Human Adipose-Derived Stem Cells Using Trehalose as the Sole Cryoprotectant. ACS applied materials & interfaces, 7(8), 5017-5028.
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4

Quantitative Glycogen Measurement Protocol

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Cells for quantitative glycogen measurement were collected as for trehalose measurements. Glycogen extraction and measurement protocols were used from (Ewald et al., 2016 (link)). 125 ul of 0.25 M sodium carbonate was added to the frozen cell pellet, which was then vortexed vigorously and incubated at 85°C for 4 hours with gentle shaking. Next, 75 ul of 1 M acetic acid, 300 ul sodium acetate (200 mM, p.H. 5.2), and 2 ul amyloglucosidase (from Aspergillus Niger, Sigma, 1000 U/ml) were added to each sample. Samples were mixed by pipetting and incubated at 60°C overnight (~16 hours). Samples were centrifuged and the supernatants filtered using spin columns to remove cellular debris. Glucose released from glycogen digestion with amyloglucosidase was measured enzymatically using the Trehalose Assay Kit (Megazyme, K-TREH) without the trehalase.
Persson L.B., Ambati V.S, & Brandman O. (2020). Cellular control of viscosity counters changes in temperature and energy availability. Cell, 183(6), 1572-1585.e16.
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5

Trehalose and Glutathione Quantification in C. elegans

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Worms were grown to second day of adulthood on standard agar plates seeded with E coli K12. The glp-4(bn2ts) and daf-2(e1370ts) mutations required a temperature switch from 16°C to 24°C at the L3 stage. Young adult worms were washed in S-basal and were frozen immediately. Samples were thawed and homogenized by bead beating as described in ref. (22 (link)). Trehalose was measured in microplates using a trehalose assay kit according to the manufacturer’s instructions (Megazyme, Wicklow, Ireland) (25 (link)). Glutathione was measured in microplates using a standard glutathione assay kit (Oxford Biomedical Research) according to the manufacturer’s instructions.
Depuydt G., Shanmugam N., Rasulova M., Dhondt I, & Braeckman B.P. (2016). Increased Protein Stability and Decreased Protein Turnover in the Caenorhabditis elegans Ins/IGF-1 daf-2 Mutant. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, 71(12), 1553-1559.
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6

Rat Hepatocyte Maintenance Protocol

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Phosphate-buffered saline tablets, HEPES buffer powder, o-phenylenediamine dihydro-chloride for rat albumin assay, thiazolyl blue tetrazolium bromide for the MTT assay, epidermal growth factor (EGF), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), glucose, sucrose, glucagon and hydrocortisone were all obtained from Sigma, St. Louis, MO. Trehalose was purchased from Ferro-Pfanstiehl, Waukegan, IL, USA; trypsin-EDTA, Dulbecco’s Modified Eagle’s medium (DMEM), fetal bovine serum (FBS) were from Life Technologies, Grand Island, NY; fibronectin was obtained from BD, Franklin Lakes, NJ. The trehalose assay kit was purchased from Megazyme, Ireland and rat albumin antibody (peroxidase-conjugated sheep IgG fraction) from MPBio, Santa Ana, CA, USA.
Phosphate-buffered saline was prepared by dissolving PBS tablets in ultra-high purity (>18 mOhm) distilled water. Collagen solution was prepared following an in-house protocol from rat tail. Rat hepatocyte maintenance medium was prepared as the following: high glucose (4.5g/L), DMEM supplemented with 10% FBS, 0.02 mg/L glucagon, 20 ng/mL EGF, 7.5 μg/mL hydrocortisone and 2% (v/v) penicillin/streptomycin.
Abazari A., Meimetis L.G., Budin G., Bale S.S., Weissleder R, & Toner M. (2015). Engineered Trehalose Permeable to Mammalian Cells. PLoS ONE, 10(6), e0130323.
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7

Enzymatic Conversion of Maltose to Trehalose

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Crude extracts of B. subtilis harboring or not haboring plasmids were incubated with maltose of different concentrations ranging from 100 mM to 1000 mM in 100 mM PBS pH 6.0-8.0 at 25-45 o C for 2 hours and then heated at 90 o C for 10 min to stop enzyme activity. Maltose will be converted to trehalose if TreS is present in the crude extract. Extracts of B. subtilis 1012, or B. subtilis 1012 harboring pHT01 vector or non-induced B. subtilis 1012 harboring pHT01-treS served as negative controls (without TreS). The amount of trehalose in the reaction product was measured by Trehalose Assay Kit (Megazyme, USA) following kit producer's instruction. One unit of TreS was defined as the amount of enzyme that catalyzes the formation of 1 mg of trehalose in one hour. The relative enzyme activity (%) was defined as the percentage of enzyme activity in the control (Ma et al., 2006) (link).
Trieu N.N., Nguyen T.Q., Nguyen H.D., Dat N.M., Long T.D., & Loan N.T.H. (2020). Expression and characterization of recombinant trehalose synthase in Bacillus subtilis<\i>. ACADEMIA JOURNAL OF BIOLOGY, 42(4).
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8

Trehalose Quantification in C. elegans

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About 1000 worms were collected, washed in M9 buffer and lysed in RIPA buffer by bead beating three times for 30 s at 6500 rpm with the samples chilled on ice between the homogenisation steps. Samples were then centrifuged 5 min at 10,000 × g, 4 °C. C. elegans were harvested and washed off plates using M9 buffer. Cleared lysate protein concentration was measured using the BCA assay kit (Thermo Fisher, Waltham, MA). Remaining cleared lysates were treated with 10 mg/ml alkaline borohydrides to reduce sugar and subsequently neutralised with 200 mM acetic acid. Next, the concentrations of trehalose were measured using the Trehalose Assay Kit (Megazyme, Wicklow, Ireland) following the manufacturer’s protocol using Tecan Infinite M200Pro with i-control software.
Beaudoin-Chabot C., Wang L., Celik C., Abdul Khalid A.T., Thalappilly S., Xu S., Koh J.H., Lim V.W., Low A.D, & Thibault G. (2022). The unfolded protein response reverses the effects of glucose on lifespan in chemically-sterilized C. elegans. Nature Communications, 13, 5889.
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9

Quantitative Trehalose Determination

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Ovarian tissue pieces underwent indicated microwave drying procedure, excess trehalose was dabbed off from the tissues with kimwipes. Tissues were snap frozen and kept in -80°C until all samples were ready for analysis, then thawed at the same time to disrupt cell membrane before trehalose extraction. Each piece was placed in a single well of a 96-well plate and trehalose was extracted by adding 200 μl of hot (80°C) distilled water to each well and shaking at 600 rpm for 15 min. Each sample was diluted 1:10 and trehalose content was quantified with a trehalose assay kit (Megazyme) following manufacturer’s instruction. The kit utilized a series of enzymatic reactions to convert trehalose into reduced nicotinamide-adenine dinucleotide phosphate, which could be measured by the absorbance at 340 nm with a BioTek ELx808 microplate reader (BioTek Instruments).
Lee P.C., Adams D.M., Amelkina O., White K.K., Amoretti L.A., Whitaker M.G, & Comizzoli P. (2019). Influence of microwave-assisted dehydration on morphological integrity and viability of cat ovarian tissues: First steps toward long-term preservation of complex biomaterials at supra-zero temperatures. PLoS ONE, 14(12), e0225440.
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10

Fungal Trehalose Extraction and Quantification

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Fungal trehalose was extracted from five-day-old liquid cultures as described previously with modifications [41 (link)]. Fungal culture was vacuum filtered and washed with distilled H2O. Two volumes of distilled H2O were added to the washed mycelia and the samples were boiled for 20 min to inactivate enzymes and release soluble sugars. The supernatant was collected by centrifugation at 12,000 rpm for 5 min. Free D-glucose was removed from the supernatant, and then a trehalose assay was performed using a Trehalose Assay Kit (Megazyme, Chicago, IL, USA) following the manufacturer’s recommendations. Three independent samples were analyzed.
Liu C., Cleckler B, & Morsy M. (2018). Development of an Expression Vector to Overexpress or Downregulate Genes in Curvularia protuberata. Journal of Fungi, 4(2), 54.
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