The interaction between NaD1 and polysaccharides was characterized by NMR using 15N labelled NaD1 with laminarin (Sigma) as a source of β-glucan or chitohexaose (Megazyme) as a source of oligosaccharide from chitin. Spectra recorded at 600 MHz on a Bruker Avance I spectrometer included 2D 15N-Heteronuclear Single Quantum Coherence (HSQC), 3D 15N-HSQC-Nuclear Overhauser Effect spectroscopy (NOESY) and 3D 15N-HSQC-Total Correlation Spectroscopy (TOCSY). Since chemical shifts are indicative of local environment, titration experiments were conducted to determine changes that occurred upon the interaction of 15N-labelled NaD1 with laminarin or chitohexaose. Aliquots of a stock solution of laminarin (146 mg/mL H2O, 110 μL Na3PO4, 110 μL D2O) were sequentially titrated into an NMR tube containing the 15N-labelled NaD1 (400 μL 15N-NaD1 4 mg/mL 90% H2O/10% D2O v/v, 50 μL 0.5 M Na3PO4, pH 6.3). For the chitohexaose experiments, a stock solution of hexaacetyl chitohexaose (3.3 mg/mL H2O) was added similarly to a sample of 15N-labelled NaD1 (450 μL 15N-NaD1 1.1 mg/mL). Chemical shift differences of greater than 0.1 ppm (for 15N shifts) and 0.01 ppm (for proton shifts) were considered significant.
Avance 1 spectrometer
Manufactured by Bruker
Sourced in United States
The Avance I spectrometer is a nuclear magnetic resonance (NMR) spectrometer designed for high-resolution analysis of chemical samples. It provides accurate and reliable measurements of molecular structures and compositions. The core function of the Avance I is to generate and detect radio frequency (RF) signals to obtain detailed NMR spectra of various materials.
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Lab products found in correlation
Laminarin, by Merck Group (1 mentions)
Chitohexaose, by Megazyme (1 mentions)
Topspin 3, by Bruker (1 mentions)
Tci cryogenic probe, by Bruker (1 mentions)
Paraffin oil, by Merck Group (1 mentions)
Avance 3 hd spectrometer, by Bruker (1 mentions)
Avance 2 700 mhz spectrometer, by Bruker (1 mentions)
Empyrean diffractometer, by Malvern Panalytical (1 mentions)
Invia confocal raman microscope, by Renishaw (1 mentions)
Topspin 2, by Bruker (1 mentions)
Paravision 5, by Bruker (1 mentions)
5 mm txi cryoprobe, by Bruker (1 mentions)
Avance 3 spectrometer, by Bruker (1 mentions)
Inova spectrometer, by Agilent Technologies (1 mentions)
11 protocols using avance 1 spectrometer
1
NMR Characterization of NaD1-Polysaccharide Interactions
2
NMR Spectroscopy of 3m-Pin1 Protein
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All 3m-Pin1 spectra were recorded on a Bruker Avance I spectrometer at 16.4 T (700.13 MHz 1H frequency) equipped with a TCI cryogenic probe (Bruker Biospin, Inc.). Sample concentrations ranged from 80 to 100 μm . The 3m-pin1 backbone assignments were confirmed using established three-dimensional HNCACB (49 (link)), HNCOCACB (50 (link)), and 2D 1H-15N HSQC (51 (link)) experiments at a nominal temperature of 295 K and comparisons with the WT-Pin1 assignments. NMR data processing used TopSpin 3.5 (Bruker Biospin) and resonance assignments made with Sparky 3 (52 ) and CARA (53 ). Amide 1H-15N CSPs were calculated using Equation 1 ,
where ΔδH and ΔδN are 1H and 15N chemical shift differences, respectively.
where ΔδH and ΔδN are 1H and 15N chemical shift differences, respectively.
3
NMR Analysis of LiTFSI-G4 Mixtures
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LiTFSI-G4 mixtures were prepared in a concentration range of 0.18–2.5
mol/kg of solvent. Herein the concentration of the samples is defined
by r = [Li+]/[O], the ratio between the
concentration of lithium cations and that of the oxygen atoms in tetraglyme,
for consistency with previous work.16 (link)1H NMR spectra were acquired on the samples to identify the
relative shifts of the resonances corresponding to protons within
CH2 and CH3 groups of the tetraglyme solvent;
additionally, 1H T1 (spin–spin
relaxation) measurements were performed using a standard inversion
recovery sequence. 1H NMR experiments were performed at
a field strength of 16.4 T using a 700 MHz Bruker Avance I spectrometer,
equipped with either a Bruker 5 mm double-resonance broadband observe
(BBO) probe or a Bruker 5 mm triple-resonance inverse (TXI) probe,
with variable-temperature control. Measurements were performed using
a Larmor frequency of 700.1 MHz. The sample temperature was fixed
at 30 °C.
mol/kg of solvent. Herein the concentration of the samples is defined
by r = [Li+]/[O], the ratio between the
concentration of lithium cations and that of the oxygen atoms in tetraglyme,
for consistency with previous work.16 (link)1H NMR spectra were acquired on the samples to identify the
relative shifts of the resonances corresponding to protons within
CH2 and CH3 groups of the tetraglyme solvent;
additionally, 1H T1 (spin–spin
relaxation) measurements were performed using a standard inversion
recovery sequence. 1H NMR experiments were performed at
a field strength of 16.4 T using a 700 MHz Bruker Avance I spectrometer,
equipped with either a Bruker 5 mm double-resonance broadband observe
(BBO) probe or a Bruker 5 mm triple-resonance inverse (TXI) probe,
with variable-temperature control. Measurements were performed using
a Larmor frequency of 700.1 MHz. The sample temperature was fixed
at 30 °C.
4
Structural Characterization of PACT-D3 Mutant
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NMR samples were prepared by dialysis into 20 mM MES pH 6.5, 50 mM NaCl, 5–10 mM TCEP followed by the addition of 10% D2O and 50 μM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS). The 2D (1H, 15N) HSQC and EXSY spectra, and 3D experiments for assignment of PACT-D3 L273R, were recorded using a Bruker Avance II 700 MHz spectrometer with a triple-resonance room temperature probe. Spectra for backbone assignment of wild-type (WT) PACT-D3 were recorded on a Bruker 600 MHz Avance II+ spectrometer with triple-resonance cryoprobe, while spectra for side-chain assignment was collected on a Bruker 800 MHz Avance III HD spectrometer with triple-resonance cryoprobe. The 13C filter-edit NOESY experiment was recorded on a 50:50 mixture of [13C,15N]- and [15N]-labelled WT PACT-D3 using a Bruker 700 MHz Avance III HD spectrometer with quadruple-resonance cryoprobe. The high pressure 2D (1H, 15N) HSQC NMR experiments were recorded using a Bruker 800 MHz Avance I spectrometer, equipped with a triple-resonance room temperature probe. The sample was inserted into a ceramic tube (rated to 2.5 kbar) and pressurized with paraffin oil (Sigma) using a high-pressure syringe pump (Daedalus Innovations LLC, PA).
5
Acquisition of 1H NMR Spectra
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Spectra were acquired
on a Bruker Avance I spectrometer equipped with a variable temperature
module (resonance frequency of 400.13 MHz for 1H) and a
QNP probe. Unless otherwise specified, for 1H experiments,
90° pulses and pulse sequence recycle times of 3 s were used.
One-dimensional 1H spectra were obtained with 32 scans
and 32 K data points and were reprocessed using MestReNova software
(v6.2). Chemical shifts were referenced with respect to the DMSO peak
(δDMSO = 2.5 ppm at 298 K).
on a Bruker Avance I spectrometer equipped with a variable temperature
module (resonance frequency of 400.13 MHz for 1H) and a
QNP probe. Unless otherwise specified, for 1H experiments,
90° pulses and pulse sequence recycle times of 3 s were used.
One-dimensional 1H spectra were obtained with 32 scans
and 32 K data points and were reprocessed using MestReNova software
(v6.2). Chemical shifts were referenced with respect to the DMSO peak
(δDMSO = 2.5 ppm at 298 K).
6
Multimodal Materials Characterization
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The X-ray diffraction (XRD) patterns were collected on a PANalytical Empyrean diffractometer with Cu Ka radiation, operated at 45 kV and 40 mA. X-ray photoelectron spectroscopy (XPS) was performed on a PHI 3056 spectrometer equipped with an Al anode source operated at 15 kV, an applied power of 350 W, and a pass energy of 93.5 eV. Raman spectroscopy was performed on a Renishaw inVia confocal Raman microscope with a 532 nm laser. N2 adsorption-desorption experiments were performed on a 3 Flex Micromeritics Setup at 77 K. 13C{1H} cross-polarization magic-angle spinning (CPMAS) experiment on a Bruker Biospin AVANCE I spectrometer, operating at 9.4 T equipped with a 4 mm double-resonance MAS probe. A MAS rate of 7.5 kHz was used. STEM imaging was performed on a probe-corrected JOEL NEOARM electron microscope at 80 kV.
7
Multimodal MRI Characterization using Bruker
8
NMR Experiments of AFABP Protein Characterization
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The two- and three-dimensional NMR experiments [14] were performed at 20 °C on a Bruker Avance I spectrometer operating at 500 MHz and equipped with a 5-mm TXI cryoprobe (Bruker Biospin, Billerica, MA). The AFABP sample (400 μM, assuming all proteins are monomers) was prepared in a solution that contained 10% (v/v) D2O, 10 mM potassium phosphate, 150 mM potassium chloride and 0.2 g/L sodium azide, adjusted to pH 7.4. The 2D 1H–15N heteronuclear single quantum correlation (HSQC) spectra were acquired with respective spectral widths of 14 ppm and 32 ppm in the 1H and 15N dimensions, requiring 8–128 scans (0.7–11 h) in separate experiments at a range of protein concentrations. For 2D nuclear Overhauser and total correlation spectroscopy (1H–15N NOESY-HSQC and 1H–15N TOCSY-HSQC) experiments, the typical mixing and spin-lock times were 150 ms and 70 ms, respectively. The triple-resonance experiments (HNCO, HN(CA)CO, HNCACB and CBCA(CO)NH) were conducted using typical acquisition and processing parameters described previously [15] (link), [16] (link). The resulting data were processed using NMRPipe software [17] (link) and analyzed by NMRViewJ software [18] (link).
9
NMR Spectroscopic Analysis of Tarp Protein Backbone
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All NMR spectra were acquired at 298 K either at 800 MHz (Bruker Avance III spectrometer equipped with a triple resonance indirect detect TXI probe with z-gradients), 500 MHz (Bruker Avance I spectrometer with a triple resonance indirect detect TXI probe with z-gradients, or 600 MHz (Varian Inova spectrometer with a 5 mm triple resonance z-gradient probe). The Tarp backbone was assigned through application of the standard “backbone-walk” methodology with the acquisition of CBCA(CO)NH39 (link), HNCACB40 (link), HNN41 (link), and [1H-15N]-TOCSY-HSQC42 spectra. All assignable Tarp chemical shifts have been deposited in the BMRB; accession code 27263. The DSS standard within each sample was used to reference the directly detected 1H dimension; all indirect dimensions were referenced according to the ratio of their heteronuclear gyromagnetic ratios and specific nuclear observation frequencies as described by Wishart et al.43 (link).
10
Starch Hydrogel Characterization by NMR
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13C Solution-state NMR spectra were acquired on a Bruker Avance I spectrometer, operating at 13C frequency of 125.79 MHz, equipped with a 5 mm probe. Hydrogels were prepared directly in Pyrex® NMR tubes (Norell Inc.®), starting with 10 % (w/v) starch/D2O suspensions with total volume of 700 μL and following all other gelatinisation and storage procedures as described above (see Hydrogel preparation section). All 1H and direct 13C detection experiments were acquired with a 10 μs 13C rf pulse, 2.0 s relaxation delay, a minimum of 2000scans and carried out at 25 °C. The short recycle delay was chosen to probe the structure of the liquid-like components in the hydrogels.
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