Conduritol b epoxide

Manufactured by LGC

Sourced in Canada

Conduritol B epoxide is a chemical compound that is commonly used in the field of organic synthesis and biochemistry. It is a cyclic derivative of the sugar alcohol inositol, and its core function is to serve as a precursor or intermediate in the synthesis of various organic compounds.

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Lab products found in correlation

Cell culture media, by Thermo Fisher Scientific (1 mentions) Diltiazem hydrochloride, by Bio-Techne (1 mentions) 4 methylumbelliferyl β d glucopyranoside mug, by Merck Group (1 mentions) Fluoroskan ascent fl, by Thermo Fisher Scientific (1 mentions) Sodium taurocholate, by Fujifilm (1 mentions) Triton x 100, by Merck Group (1 mentions) 4 mug, by Merck Group (1 mentions) Sodium taurodeoxycholate, by Merck Group (1 mentions) Spectramax m2, by Molecular Devices (1 mentions)

5 protocols using conduritol b epoxide

Fluorescent Glucoside Assay Protocol

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4-Methylumbelliferyl β-D-glucopyranoside (MUG) was from Sigma (St. Louis, MO). Conduritol B epoxide (CBE) was from Toronto Research Chemicals (Downsview, ON, Canada). Diltiazem hydrochloride was from Tocris Bioscience (Ellisville, MO). Cell culture media were purchased from Gibco (Grand Island, NY).

Tan Y.L., Genereux J.C., Pankow S., Aerts J.M., Yates JR I.I.I, & Kelly J.W. (2014). ERdj3 is an Endoplasmic Reticulum Degradation Factor for Mutant Glucocerebrosidase Variants Linked to Gaucher’s Disease. Chemistry & biology, 21(8), 967-976.

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Assay for GCase Enzymatic Activity

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The assay for GCase enzymatic activity was performed as described[11 (link)]. Medaka brains were homogenized in 40 μl sample buffer (10 mM Tris-HCl, 150 mM NaCl, 1% (v/v) Triton X-100, pH 7.4), sonicated, and centrifuged at 10,000 ×g at 4°C for 5 min. Aliquots containing 50 μg protein were incubated in assay buffer (5 mM 4-Methylumbelliferyl β-d-glucopyranoside (Wako, #324–37441), 1% (w/v) sodium taurocholate (Wako, #197–10033), 50 mM sodium citrate, 50 mM sodium phosphate, pH 5.0) in the presence or absence of 2 mM Conduritol B epoxide (Toronto Research Chemicals, #C666000) in a total volume of 100 μl at 37°C for 4 hr. The reaction was stopped by adding 100 μl of 0.4 M glycine, pH 10.8, and the fluorescence at 460 nm (emission 355 nm) was measured with Fluoroskan Ascent FL (Thermo Fisher).

Uemura N., Koike M., Ansai S., Kinoshita M., Ishikawa-Fujiwara T., Matsui H., Naruse K., Sakamoto N., Uchiyama Y., Todo T., Takeda S., Yamakado H, & Takahashi R. (2015). Viable Neuronopathic Gaucher Disease Model in Medaka (Oryzias latipes) Displays Axonal Accumulation of Alpha-Synuclein. PLoS Genetics, 11(4), e1005065.

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Measuring GBA Activity in DBS

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GBA activity was measured as previously described [20 (link)] Briefly, one 3.2 mm-diameter punch from each DBS sample was extracted in 200 ;L 0.2 M citrate phosphate buffer, pH 5.2 containing 1% Triton^® X-100 (Sigma, St. Louis, MO) and 1% sodium taurodeoxycholate (≥97% purity, Sigma, St. Louis MO) in a 96-well plate. Substrate solution (12.5 mM 4-MUG, Sigma, St. Louis, MO) was prepared either with or without inhibitor (0.5 mM Conduritol B Epoxide, Toronto Research Chemicals). Inhibited or uninhibited substrate solution was mixed 2:1 with DBS extract and incubated for 20 h at 37 °C. To stop the reactions, 100 μL of 0.5 M EDTA (pH 11.5) was added to each well. An eight point 4-MU standard curve (0 – 0.67 μM) was prepared on each plate in duplicate. The plate was read in a fluorometer at 355 nm excitation and 460 nm emission wavelengths. GBA activity was determined by subtracting the background activity measured in the inhibited reaction from that in the uninhibited reaction. Disease cut-off (1.71 μmol/L/h) was established as described in methods section 2.6. The limit of blank (LOB=0.16 μmol/L/h) and limit of detection (LOD=0.41 μmol/L/h) for the fluorescence assay were established previously [20 (link)].

Wolf P., Alcalay R.N., Liong C., Cullen E., Paciulo M.W., Nichols W.C., Gan-Or Z., Chung W.K., Faulkner T., Bentis C., Pomponio R.J., Ma X., Zhang X.K., Keutzer J.M, & Oliva P. (2017). Tandem mass spectrometry assay of β-Glucocerebrosidase activity in dried blood spots eliminates false positives detected in fluorescence assay. Molecular genetics and metabolism, 123(2), 135-139.

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Quantifying Lysosomal Hydrolase Activities

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Glucocerebrosidase and hexosaminidase activities were determined using the artificial substrates 4-methylumbelliferyl (4-MU)-β-D-glucoside or 4-MU-N-acetyl-β-D-glucosaminide as previously described (31). Briefly, twenty bodies without heads or 60 heads of 5-7-day-old flies were homogenized in 300 ul of KP buffer (50 mM potassium phosphate buffer, 0.25% Triton X-100, pH 6.5) and centrifuged at 4,000 g for 5 minutes. Supernatants were transferred to a fresh tube and assayed for the hydrolase activity in 0.1M sodium acetate buffer (pH 4.5). Lysates were incubated with Conduritol B epoxide (50 μM, 45 minutes, Toronto Research Chemicals, Inc) to subtract for non-lysosomal glucocerebrosidase mediated activity. Hydrolase reactions were stopped (1M Glycine-NaOH buffer, pH 12.5) and fluorescence quantified using a Spectra Max M2 fluorescent plate reader (excitation 365 nm, emission 445 nm, Molecular Devices). Protein levels were determined using the micro-BCA kit (Pierce, Rockford, IL). Each experiment was performed at least three times.

Davis M.Y., Trinh K., Thomas R.E., Yu S., Germanos A.A., Whitley B.N., Sardi S.P., Montine T.J, & Pallanck L.J. (2016). Glucocerebrosidase Deficiency in Drosophila Results in α-Synuclein-Independent Protein Aggregation and Neurodegeneration. PLoS Genetics, 12(3), e1005944.

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Quantifying Glucocerebrosidase Activity Assay

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GCase enzyme activity was determined using a 0.1 M acetate assay buffer pH 5.0 containing 3 mM 4-MUG (4-methylumbelliferyl β-D-glucopyranoside), 0.15% (v/v) Triton X-100, and 0.15% (w/v) taurodeoxycholic acid sodium salt (Calbiochem). Purified GCase or cell lysate was added to the activity assay buffer and incubated at 37 °C for 1 h. The reaction was stopped with the addition of a 2× volume of 0.2 M glycine buffer pH 10.8. Product 4-methylumbelliferone was detected using the Victor 3 fluorimeter (filter set excitation = 355 nm, emission = 460 nm). Background β-glucosidase (GBA2) enzyme activity was subtracted from lysate activity using duplicate controls containing 1 μM conduritol B epoxide (Toronto Research Chemicals Inc.) added to enzyme reactions to inhibit GCase activity. Black 96-well plates were typically used although limited initial work was performed with white 96-well plates. The 4-methylumbelliferone detection is highly linear between 10nM–10 μM in the black 96-well plate and between 10nm–5 μM in the white 96-well plate. Enzyme activity units are defined as nmol 4-methylumbelliferone released per hour.

Gramlich P.A., Westbroek W., Feldman R.A., Awad O., Mello N., Remington M.P., Sun Y., Zhang W., Sidransky E., Betenbaugh M.J, & Fishman P.S. (2016). A peptide-linked recombinant glucocerebrosidase for targeted neuronal delivery: Design, production, and assessment. Journal of biotechnology, 221, 1-12.

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