Microwave reactor

Manufactured by CEM Corporation

Sourced in United States

The Microwave reactor is a laboratory equipment used for performing chemical reactions under microwave irradiation. It provides a controlled environment for rapid heating and precise temperature regulation during the reaction process.

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Lab products found in correlation

Peptide n glycosidase f, by New England Biolabs (4 mentions) Spe plate, by Grace Bio-Labs (2 mentions) Pngase f, by CEM Corporation (2 mentions) Pngase f, by New England Biolabs (1 mentions) Nh4 2mos4, by Merck Group (1 mentions) N n dimethylformamide (dmf), by Merck Group (1 mentions)

9 protocols using microwave reactor

N-Glycan Extraction and Purification

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Enzymatic release and solid-phase extraction of N-glycans were performed according to optimized procedures published by Kronewitter et al.^{26 (link)} Briefly, membrane glycoproteins were denatured by rapid thermal cycling (25–100 °C) in an aqueous solution of 100 mM ammonium bicarbonate and 5 mM dithiothreitol. Next, 2.0 μL (or 1000 U) of peptide N-glycosidase F (New England Biolabs) were added and the mixture was incubated in a microwave reactor (CEM Corporation) for 10 minutes at 20 watts. Following the addition of 800 μL of cold ethanol, the mixture was chilled at −80 °C for 1 hour, then centrifuged in order to precipitate out the deglycosylated proteins. The glycan-rich supernatant fraction was collected and dried in vacuo.
Graphitized carbon solid-phase extraction was performed using an automated liquid handler (Gilson). Graphitized carbon cartridges (150 mg, 4.0 mL, Grace Davison) were washed with 80% acetonitrile / 0.10% trifluoroacetic acid (v/v) in water, then conditioned with pure water. Aqueous N-glycan solutions were loaded onto the cartridges and washed with pure water to remove salts and buffer. Membrane N-glycans were eluted with sequential addition of 10% acetonitrile, 20% acetonitrile, and 40% acetonitrile / 0.05% trifluoroacetic acid (v/v) in water. Samples were dried in vacuo.

Hua S., Saunders M., Dimapasoc L.M., Jeong S.H., Kim B.J., Kim S., So M., Lee K.S., Kim J.H., Lam K.S., Lebrilla C.B, & An H.J. (2014). Differentiation of Cancer Cell Origin and Molecular Subtype by Plasma Membrane N-Glycan Profiling. Journal of proteome research, 13(2), 961-968.

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Glycomic Sample Preparation and N-Glycan Isolation

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Details of the glycomic sample preparation have been described previously ((Wu et al., 2010 (link); Wu et al., 2011 (link))). Extracted cell membrane fractions or protein (RNase B) were suspended with 100 μl of 100 mM NH₄HCO₃ in 5 mM dithiothreitol and heated in boiling water for 2 min to denature the proteins. Solutions of with 2 μl of peptide N-glycosidase F (New England Biolabs, MA, United States) were added to the samples, and the resulting solutions were then incubated in a microwave reactor (CEM Corporation, NC, United States) at 20 W, 37°C for 10 min. The samples were further placed in a 37°C water bath for 18 h. Ultracentrifugation at 200,000 × g for 45 min was performed to precipitate proteins, and the supernatant containing N-glycans was collected and desalted using porous graphitic carbon (PGC) on a 96-well SPE plate (Grace, IL, United States). The plate was equilibrated with 80% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid. Then the samples were loaded onto the plate and washed with nanopure water. N-Glycans were eluted with a solution of 40% (v/v) acetonitrile containing 0.05% (v/v) trifluoroacetic acid, and dried in vacuo using miVac (SP Scientific, PA, United States) prior to further analysis.

Sheng Y., Vinjamuri A., Alvarez M.R., Xie Y., McGrath M., Chen S., Barboza M., Frieman M, & Lebrilla C.B. (2022). Host Cell Glycocalyx Remodeling Reveals SARS-CoV-2 Spike Protein Glycomic Binding Sites. Frontiers in Molecular Biosciences, 9, 799703.

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N-Glycan Extraction and Purification

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Proteins were suspended with 100 μL of 100 mM NH₄HCO₃ in 5 mM dithiothreitol and heated at 100 °C for 10 s to thermally denature the proteins. To release the glycans, 2 μL of peptide N-glycosidase F (New England Biolabs, MA) were added to the samples, which were then incubated in a microwave reactor (CEM Corporation, NC) at 20 watts for 10 min. After addition of 400 μL of cold ethanol, samples were incubated at –80 °C for 1.5 h to precipitate deglycosylated proteins and centrifuged at 21 000 × g for 20 min. The precipitated proteins were dried and stored at –20 °C for O-glycan preparation and analysis. The supernatant containing N-glycans was collected and dried. The released N-glycans were purified by solid-phase extraction using porous graphitized carbon (PGC) packed cartridges (Grace, IL). The cartridges were first equilibrated with nanopure water and a solution of 80% (v/v) acetonitrile and 0.05% (v/v) trifluoroacetic acid in water. The dried samples were solubilized, loaded onto the cartridge, and washed with nanopure water at a flow rate of 1 mL min^–1 to remove salts and buffer. N-Glycans were eluted with a solution of 40% (v/v) acetonitrile and 0.05% (v/v) trifluoroacetic acid in water and dried prior to mass spectrometric analysis.

Park D.D., Xu G., Wong M., Phoomak C., Liu M., Haigh N.E., Wongkham S., Yang P., Maverakis E, & Lebrilla C.B. (2018). Membrane glycomics reveal heterogeneity and quantitative distribution of cell surface sialylation †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc01875h. Chemical Science, 9(29), 6271-6285.

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N-Glycan Profiling from Membrane Fractions

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The membrane fraction was harvested according to the standard procedure53 (link) with minor modification. In brief, CCA cells were homogenized in 20 mM HEPES-KOH, pH 7.4 containing 0.25 M sucrose and 1:100 protease inhibitor, using probe sonication (25 Amplitude, pulsed on 5 sec and off 10 sec for 5 times). Cell nuclei were fractionated by centrifugation at 2,000 xg, for 10 min. Then, the membrane fraction was separated by 3 times of ultra-centrifugation at 200,000 xg, 45 min. The final pellet was collected as the membrane fraction and total N-glycans were released using 1,000 U of PNGase-F at 37°C in a microwave reactor (CEM Corporation, Matthews, NC) at 20 watts for 10 min. The released N-glycans were enriched and purified by porous graphitized carbon solid-phase extraction as described previously.48 (link) The unbound fraction was first eluted with 9 ml of pure water and the bound N-glycan fraction was eluted with 4 ml of 40% ACN and 0.1% TFA in pure water.

Phoomak C., Silsirivanit A., Park D., Sawanyawisuth K., Vaeteewoottacharn K., Wongkham C., Lam E.W., Pairojkul C., Lebrilla C.B, & Wongkham S. (2018). O-GlcNAcylation mediates metastasis of cholangiocarcinoma through FOXO3 and MAN1A1. Oncogene, 37(42), 5648-5665.

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N-Glycan Release and Purification

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Membrane pellets were resuspended in 100 μL of 100 mM ammonium bicarbonate with 5 mM dithiothreitol and heated for 10 s at 100 °C to thermally denature the proteins. To release the N-glycans from membrane proteins, 2 μL of peptide N-glycosidase F (New England Biolabs, MA) were added to the samples and incubated at 37 °C in a microwave reactor (CEM Corporation, NC) for 10 min at 20 watts. After addition of 350 μL of nanopore water, samples were ultracentrifuged at 200 000×g for 45 min at 4 °C to precipitate the membrane fractions with lipids and residual deglycosylated proteins. The supernatant containing the released N-glycans was collected. N-Glycans were purified by solid phase extraction containing a porous graphitized carbon (PGC) matrix (Grace, IL). Eluted fractions were dried in vacuo before they were reconstituted in 30 μL of nanopore water and analyzed by LC-MS/MS.

Xu G., Wong M., Li Q., Park D., Cheng Z, & Lebrilla C.B. (2019). Unveiling the metabolic fate of monosaccharides in cell membranes with glycomic and glycoproteomic analyses †Electronic supplementary information (ESI) available: Supplementary information includes six figures. See DOI: 10.1039/c9sc01653h. Chemical Science, 10(29), 6992-7002.

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Synthesis of Zinc Oxide Nanoparticles

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In a typical synthesis, aqueous solution of zinc acetate dihydrate (10 mL) with certain molarity was mixed with an equal volume of 0.9 M NaOH solution. The mixture was subsequently exposed to irradiation in a microwave reactor (CEM Corporation, USA) for 20 minutes at a constant temperature of 80 °C with a maximum permissible power of 100 W. During the synthesis process, 0.035 M, 0.07 M, 0.14 M, 0.28 M, and 0.56 M solutions of zinc acetate dihydrate were employed, and samples derived from these solutions were denoted as 0.035-ZnO, 0.07-ZnO, 0.14-ZnO, 0.28-ZnO, and 0.56-ZnO, correlating to their respective Zn precursor molar concentrations. Higher molar concentrations were not studied here due to solubility limit restrictions. The formed milky-white suspensions were centrifuged at 5000 rpm for 2 minutes, washed several times, and then dried at 60 °C for 24 h.

Rustembekkyzy K., Sabyr M., Kanafin Y.N., Khamkhash L, & Atabaev T.S. (2024). Microwave-assisted synthesis of ZnO structures for effective degradation of methylene blue dye under solar light illumination. RSC Advances, 14(23), 16293-16299.

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N-Glycan Extraction from Membrane Fraction

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The membrane fraction was harvested according to the standard procedure [53 (link)] with minor modification. In brief, CCA cells were homogenized in 20 mM HEPES-KOH, pH 7.4 containing 0.25 M sucrose and 1:100 protease inhibitor, using probe sonication (25 Amplitude, pulsed on 5 s and off 10 s for 5 times). Cell nuclei were fractionated by centrifugation at 2000 × g, for 10 min. Then, the membrane fraction was separated by 3 times of ultra-centrifugation at 200,000 × g, 45 min. The final pellet was collected as the membrane fraction and total N-glycans were released using 1000 U of PNGase-F at 37 °C in a microwave reactor (CEM Corporation, Matthews, NC) at 20 watts for 10 min. The released N-glycans were enriched and purified by porous graphitized carbon solid-phase extraction as described previously [48 (link)]. The unbound fraction was first eluted with 9 ml of pure water and the bound N-glycan fraction was eluted with 4 ml of 40% ACN and 0.1% TFA in pure water.

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N-Glycan Release and Purification

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Plasma membrane pellets were resuspended with 100 μL of 5 mM DTT in 100 mM NH₄HCO₃ and heated in a 100 °C water bath for 1 min to thermally denature the proteins. The cleavage of N-glycans was performed by adding 2 μL of peptide N-glycosidase F (PNGase F, New England Biolabs, MA) followed by the incubation at 37 °C in a microwave reactor (CEM Corporation, NC) for 10 min at 20 watts. To convert the N-glycans from amines to aldehydes, the sample was allowed to stand in a 37 °C water bath overnight. To separate the released N-glycans from the membrane fractions, residual deglycosylated proteins and lipids, 350 μL of nanopure water was added and ultra-centrifugation at 200 000 × g was conducted for 45 min. The released N-glycans contained in the supernatant were purified using porous graphitic carbon (PGC) on an SPE plate (Grace, IL) and eluted with 40% (v/v) ACN and 0.5% (v/v) TFA in water. The eluted samples were dried in vacuo and stored at –20 °C until analysis.

Li Q., Xie Y., Xu G, & Lebrilla C.B. (2019). Identification of potential sialic acid binding proteins on cell membranes by proximity chemical labeling †Electronic supplementary information (ESI) available. See DOI: 10.1039/c9sc01360a. Chemical Science, 10(24), 6199-6209.

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Synthesis of Ammonium Molybdenum Sulfide Nanoparticles

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All chemicals were used as received without further purification. In a typical synthesis, 10 mg (NH₄)₂MoS₄ (99.97%, Sigma-Aldrich) was added to 6 ml DMF (anhydrous, 99.8%, Sigma-Aldrich) followed by stirring for 15 min under ambient condition. The resulting solution was transferred into a 10 ml microwave reaction vessel, which was then heated to 240 °C at the fast ramp and the temperature was maintained for 2 h in a microwave reactor (CEM corporation) operated under the sealed vessel mode. The reaction solution was cooled to room temperature with pressurized nitrogen flow and the resulting black product was collected via centrifugation (8,000 r.p.m. for 5 min). The precipitate was washed with distilled water and absolute ethanol for at least four times to remove ions and possible remnants, followed by drying at 60 °C in an oven for 4 h. For the time-dependent experiment, the temperature was set at 240 °C while reactions stopped at different times; for the temperature-dependent experiment, the reaction time was 2 h while the reaction temperatures were altered intentionally, all the corresponding products were collected for further studies.

Gao M.R., Chan M.K, & Sun Y. (2015). Edge-terminated molybdenum disulfide with a 9.4-Å interlayer spacing for electrochemical hydrogen production. Nature Communications, 6, 7493.

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