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35 protocols using glovebox

1

Synthesis of Functionalized Polyether Macromonomers

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All glassware used for the polymerisations was dried overnight at 120 °C prior to use. Potassium tert butoxide (KOtBu, ≥98%), 1,4,7,10,13,16-hexaoxacyclooctadecane (18-crown-6, ≥99%), 1,2-butylene oxide (BO, 99%), ethyl vinyl ether (EVE, ≥98%), glycidol (96%), calcium hydride (CaH2, 95%), potassium (K, chunks in mineral oil, 98%) and naphthalene (Naph, 99%) were purchased from Sigma-Aldrich (Switzerland) and used as received. Dry THF was obtained from an inert solvent purification system PureSolv MD 5 (Inert Technology, USA), dry 1,4-dioxane was purchased from Acros Organics (Belgium). The chemicals were stored under argon in a glovebox (MBraun Labstar, Germany). All other solvents used were in HPLC grade and purchased from JT Baker (USA), VWR (Switzerland) or Scharlau (Germany). Deionised water was obtained from a Milli-Q Q-POD device (Merck, Germany). Dulbecco's PBS buffer was purchased from Bioconcept Ltd (Switzerland).
Potassium naphthalenide (KNaph) was prepared by adding potassium (1.19 g, 30.5 mmol, 1 eq.) to a stirred solution of naphthalene (4.11 g, 32.0 mmol, 1.05 eq.) in dry THF (61 mL, 0.5 mol L−1) under argon. EEGE was synthesised following the standard protocol49 (link) from glycidol and EVE and dried over CaH2. The synthesis protocol and the 1H-NMR spectrum (Fig. S1) can be found in the ESI.
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2

Recombinant AsqJ Enzyme Production

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Plasmids (pET-28a) carrying the Aspergillus nidulans AsqJ gene were synthesized by the commercial GeneArt Gene Synthesis service (Invitrogen). The plasmids were transformed into BL21 (DE3) E. coli cells (New England Biolabs) and cultured in LB medium. Overexpression was induced by Isopropyl β-d-1-thiogalactopyranoside (GeminiBio) at 27 °C for 4 hours before cells were harvested. The enzyme was purified using a Ni-NTA agarose column, and excess salt and protein-bound metal ions were removed by dialysis. Oxygen was removed from the concentrated protein solution by degassing on a Schlenk line and equilibrating in a glove box (MBraun). The protein stock was aliquoted and stored at −80 °C. Details of the experimental conditions are provided in the Supporting Information.
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3

Coin Cell Assembly for LiCoO2 Cathode

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All compounds used were dried to avoid HF formation in the electrolyte and were assembled in a glove box under argon (MBraun, Germany) having <0.1 ppm of water and oxygen. Typically, the LiCoO2 electrode was assembled in a coin cell using lithium metal as anode, a few drops of an ethyl carbonate (EC) and diethylene carbonate (DEC) mixture in a 1:1 volume ratio with 1 M LiPF6 and 2 wt% of vinylene carbonate as electrolyte with respect to solvents and LiPF6 as well as a Celgard separator.
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4

Synthesis of Alkylated Phenols and Amines

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Solvents for synthesis were purified by standard methods: THF [high-performance
liquid chromatography (HPLC), VWR] was distilled from Na/benzophenone,
MeOH (HPLC, VWR) was distilled, and DCM (99.8% VWR) was distilled
from P2O5, n-hexane (HPLC,
VWR). Solvents for a standard workup and chromatography were used
as received. All chemicals were obtained from commercial sources and
used without further purification: 2,4-tert-buthylphenol
(99%, Sigma-Aldrich), 4-tert-buthylphenol (99%, Sigma-Aldrich), N-methylcyclohexylamine (99%, Sigma-Aldrich) N-methyldodecylamine (98%, Alfa Aesar), formaldehyde (37% solution
in H2O, Sigma-Aldrich), and sodium hydride (95%, Sigma-Aldrich).
The 1H, 13C NMR spectra were obtained using
a Bruker AVANCE 500 MHz spectrometer. The chemical shifts are given
in ppm relative to the residual signals of the solvent (CDCl3, 1H: 7.26 ppm, 13C: 77.16 ppm). HRMS spectra
were recorded using Bruker MicOTOF-Q spectrometers with Supporting Information ion source and time-of-flight
mass analyzer. Microanalyses were conducted with an Elementar CHNS
Vario EL III analyzer. All the reactions and operations which required
an inert atmosphere of N2 were performed by using a glovebox
(MBraun) or standard Schlenk apparatus and vacuum line techniques.
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5

Circular Dichroism Analysis of Zn(II) Peptide

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Circular dichroism (CD) measurements were performed at 24°C with an Olis RSM 1000 CD spectrometer using a quartz 1 mm pathlength gas-tight cuvette. CD samples were prepared in 10 mM tris(hydroxymethyl)aminomethane buffer, pH 7.5 for a final peptide concentration of 20 μM within a glovebox (MBraun), then sealed with a septum to retain an anaerobic environment during data collection. To test for the displacement of Zn(II) with silver species, the following were added to samples of 20 μM Zn(II) bound peptide, then allowed to react for 20 min prior to taking a spectrum: 44 μM AgNO3, 6.7 nM 10 nm AgENMs, or 0.42 nM 40 nm AgENMs. Three scans were averaged per sample.
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6

Electrochemical Characterization of CyGly

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All electrochemical experiments, electrode modifications and handling of the CyGly were carried out inside a glovebox (MBRAUN) filled with nitrogen. A set of mass flow-controllers (Brooks Instruments) were used to control the gas composition flushed through the electrochemical cell. The total flow in all experiments was 1000 mL min−1, unless stated otherwise. An oxygen filter (Air Liquide) before the electrochemical cell avoids any undesired O2 contamination. The potential was controlled by a VersaSTAT 4-400 potentiostat. A standard three electrode water jacketed electrochemical cell was used for the measurements with a Pt wire as counter electrode and a saturated calomel electrode located in a side arm as reference electrode. All potentials were converted to the standard hydrogen electrode (SHE) by adding + 241 mV.
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7

Electrochemical Performance Assessment of ZnO/NCNT Anodes

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A two-electrode CR2025 coin cells were assembled in a glovebox (MBraun) filled with argon and employed to assess the electrochemical performance of the samples. The working electrodes were obtained by a slurry coating process. The slurries were formed by mixing active materials (ZnO/NCNT or ZnO/CNT), carbon black (CB), and polyvinylidene fluoride (PVDF) with a mass ratio of 80:10:10 in N-methyl-2-pyrrolidine (NMP). The resulting homogenously slurries were coated on copper foil which served as current collector and dried at 60 °C under a vacuum oven for 12 h to remove solvent and water. Then the working electrodes were cut with 1.5 cm diameter and a mass loading of ~0.85 mg cm−2 as the anode. The pure lithium metal foil was used as reference electrode. The polypropylene micro-porous film (Cellgard 2300) was used as a separator. The electrolyte was 1.0 M LiPF6 dissolved in a mixture of ethylene carbonate/diethylcarbonate/dimethylcarbonate (EC/DEC/DMC) (1:1:1 by volume). Galvanostatic charge–discharge tests were detected on NEWARE battery test system (Shenzhen, China) with a potential range from 0.005 and 3.0 V vs. Li/Li+, at a wide range of current rates.
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8

Synthesis and Purification of Functionalized PPDL

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All chemicals were obtained as reagent grade from Sigma-Aldrich (St. Louis, MO, USA), Carl Roth (Karlsruhe, Germany) or Alfa Aeser (Haverhill, MA, USA) and used as received. High performance liquid chromatography (HPLC) grade solvents were purchased dry from Carl Roth. Freshly distilled chloroform and toluene were stored over molecular sieves in the glovebox. 2,3-Dihydro-5H-1,4-benzodioxepin-5-one (2,3-DHB) and pentadecalactone (PDL) were sublimated and dried under high vacuum for three days. Propargyl alcohol (PA) and benzyl alcohol (BA) were dried over calcium hydride and distilled under reduced pressure. 3-azidopropanol (AP) was synthesized according to literature procedures and was distilled under reduced pressure as well.[43 ] Triazabicyclodecene (TBD) was recrystallized from diethyl ether and dried under high vacuum for three days. Toluene, chloroform, 2,3-DHB, PDL, AP, PA, BA and TBD were stored and handled under nitrogen in a glovebox (MBRAUN, Garching, Germany). Benzyl- and alkyne-functionalized poly(pentadecalactone) (PPDL) was synthesized according to the literature.[25 ]
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9

Electrochemical Characterization of Protein Films

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All potential
measurements were performed in a glovebox (MBraun) with a PGSTAT 12
potentiostat (EcoChemie). A three-electrode configuration, including
a standard calomel reference electrode, a platinum counter electrode,
and a pyrolytic graphite edge (PGE) working electrode, was used in
conjunction with an electrochemical cell. The cell was water jacketed
and connected to a circulator for temperature control. In this study,
the temperature was maintained at 25 °C for all experiments.
In each experiment, the PGE working electrode was sanded, polished
with 1.0 μm alumina slurry, and sonicated for >10 min before
use. Protein films were generated by painting the graphite surface
of the working electrode with droplets of a concentrated protein solution
(4.6 mM, usually 2–5 μL). The working electrode was subsequently
placed in the protein-free buffer solution of the electrochemical
cell and subjected to the cycling of applied potentials. All buffers
were prepared anaerobically, with sodium acetate (5 mM), MES (5 mM),
MOPS (5 mM), TAPS (5 mM), CHES (5 mM), CAPS (5 mM), and sodium chloride
(150 mM), and adjusted to a pH range of 4.5–9.5. The raw voltammograms
were analyzed with SOAS.38 (link)
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10

Inert Atmosphere Schlenk Synthesis

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In all of the reactions in which air-sensitive chemicals were used, the reagents and solvents were dried prior to use. Diethyl ether, triethylamine, and n-hexane were distilled from Na/Ph2CO. EtOH and iPrOH were distilled from calcium hydride. Other starting materials were purchased as reagent grade and were used without further purification. Glassware was flame-dried with nitrogen or argon flushing prior to use. All of the manipulations were performed using the standard Schlenk techniques in nitrogen or argon atmosphere and using a glove box (MBraun, Garching, Germany).
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