The MALDI-TOF MS spectra were recorded on a Bruker Reflex III instrument (Germany) equipped with a nitrogen laser (337 nm), and the duration of the laser pulse was 3 ns. The parameters for positive-mode spectra in the reflection and linear modes were set according to the previous study.27 (link) In the positive reflection mode, an accelerating voltage of 20.0 kV and a reflection voltage of 23.0 kV were used. The sample solutions (10 mg mL−1 30% acetone aqueous solution) were mixed with the matrix solution (2,5-dihydroxybenzoic acid, DHB, 10 mg mL−1 30% acetone aqueous solution) at a volumetric ratio of 1 : 3. NaCl (0.52 mg mL−1) was mixed with the analyte-matrix solution (1 : 3, v/v) to strongly favor the formation of molecular ions with Na+ and avoid any misinterpretation of the mass spectra due to the formation of molecular ions with different cations. Then, the mixture (1 μL) was spotted on the steel target.
Reflex 3
Manufactured by Bruker
Sourced in Germany
The Reflex III is a versatile and high-performance X-ray diffractometer designed for a wide range of applications in materials science and research. It offers advanced features and capabilities for accurate and reliable data acquisition and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Amberlite irp 64 cation exchange resin, by Merck Group (2 mentions)
Ribo β cyanoethyl phosphoramidites, by Glen Research (2 mentions)
Pageblue, by Thermo Fisher Scientific (2 mentions)
C4 column, by Waters Corporation (2 mentions)
Ultrahydrogel 250, by Waters Corporation (1 mentions)
Ultrahydrogel 1000, by Waters Corporation (1 mentions)
Hplc system, by Waters Corporation (1 mentions)
Dhr 2 controlled stress rheometer, by TA Instruments (1 mentions)
Vario el 3, by Elementar (1 mentions)
Pgex 6p, by GE Healthcare (1 mentions)
Polyinosinic polycytidylic acid poly 1 c, by Merck Group (1 mentions)
Peptide standard 2, by Bruker (1 mentions)
α cyano 4 hydroxycinnamic acid matrix, by Bruker (1 mentions)
Amx 500, by Bruker (1 mentions)
Vario el system, by Elementar (1 mentions)
Amx 400, by Bruker (1 mentions)
Lct premier, by Waters Corporation (1 mentions)
Expedite 8909, by Thermo Fisher Scientific (1 mentions)
Mascot software, by Matrix Science (1 mentions)
Element 2, by Thermo Fisher Scientific (1 mentions)
26 protocols using reflex 3
1
MALDI-TOF Mass Spectrometry of Analytes
2
MALDI-TOF MS Protocol for Cesium Adducts
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The MALDI-TOF MS spectra were recorded on Bruker Reflex III (Germany). The experiment was carried out with reference to the method described by Xiang et al. (2006) [32] . 2,5-Dihydroxybenzoic acid (DHB, 10 mg/ml 30% acetone solution) was used as matrix. Amberlite IRP-64 cation-exchange resin (Sigma-Aldrich, USA), equilibrated in deionized water, was used to deionize the analyte, matrix, and cesium chloride solution. The sample solutions (10 mg/ml 30% acetone solution) were mixed with the cesium chloride (1.52 mg/ml) solution at a volumetric ratio of 1∶1 to promote the formation of a single type of ion adduct ([M+Cs]+). Next, the mixture was mixed with the matrix solution (1∶3, v/v). Then, the mixture (1 μl) was spotted to the steel target.
3
Characterization of PCL-b-PEG-b-PCL Triblock Copolymers
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1H NMR spectra were obtained on a DMX 400 MHz spectrometer with tetramethylsilane (TMS) as the internal standard and CDCl3 as the solvent. Size exclusion chromatography (SEC) was performed in 0.1 M NaNO3 at 40°C with an elution rate of 1.0 mL/min on a Waters HPLC system with a G1310A pump and a 2414 refractive index (RI) detector, Ultrahydrogel 250 (Waters), and Ultrahydrogel 1000 (Waters) columns in series were calibrated by polyethylene glycol standard. For triblock copolymer PCL-b-PEG-b-PCL, its SEC test was performed in THF at 35°C with an elution rate of 1 ml/min through a 7.8 × 300 mm column (with guard column) and a 2414 RI detector with polystyrene (PS) standard. MALDI-TOF MS spectra were recorded using Bruker REFLEX III. α-Cyano-4-hydroxycinnamic acid (CHCA) was used as the matrix for the ionization operated in the reflection mode. The element analysis of the product was recorded on an Elementar Vario EL-III (Germany). Rheological measurements were performed on a DHR-2 controlled stress rheometer (TA instruments, USA) with a parallel plate geometry (diameter = 8 mm). The strain was kept within the limits of the linear viscoelastic regime at a temperature of 25°C. The rubber-elastic plateau was determined from the frequency independent regime of the storage modulus.
4
Affinity Purification and Mass Spectrometry of GST-Nax
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GST-Nax is a GST fusion protein at the C-terminus (amino acid residues 1489–1681) of mouse Nax (GenBank accession no. NM_009135). pGEX-Nax was prepared by subcloning Nax cDNA from pTRE-mNax [14 (link)] into pGEX-6P (GE Healthcare) to express GST-Nax. The GST-Nax protein was expressed in the E. coli strain BL21, and purified by glutathione affinity chromatography as described previously [15 (link)].
In pull-down experiments, glutathione Sepharose beads (20 μl) were coated with GST fusion proteins (2 μg), and then incubated overnight at 4°C with synaptosomal lysate (200 μg protein) prepared from the adult rat cerebrum, as described previously [17 (link)]. After washing the beads, the bound proteins were solubilized, separated by SDS-PAGE, and stained with Coomassie Brilliant Blue. Specific bands were excised, subjected to in-gel tryptic digestion, and then applied to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Reflex III, Bruker Daltonics). Peptide mass fingerprinting was performed by a Mascot search (http://www.matrixscience.com/ ) against the NCBI nonredundant protein database.
In pull-down experiments, glutathione Sepharose beads (20 μl) were coated with GST fusion proteins (2 μg), and then incubated overnight at 4°C with synaptosomal lysate (200 μg protein) prepared from the adult rat cerebrum, as described previously [17 (link)]. After washing the beads, the bound proteins were solubilized, separated by SDS-PAGE, and stained with Coomassie Brilliant Blue. Specific bands were excised, subjected to in-gel tryptic digestion, and then applied to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Reflex III, Bruker Daltonics). Peptide mass fingerprinting was performed by a Mascot search (
5
PARP3-Mediated DNA Gap Repair Assay
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The reaction was carried out in 3 mL of HDB buffer with 2 mM MgCl2. The mixture, which contained 1 µM DNA substrate gap1, 5 µM PARP3 and 1 mM NAD+, was incubated at 37 °C for 45 min, after which it was heated at 80 °C for 5 min and loaded onto a 150 µL DEAE-toyoperl (Sigma-Aldrich, USA), at 4 °C for 8 h. After washing 3 times with 300 µL 10 mM Tris-HCl, pH 8.0, the DNA was eluted with 3 aliquots of 60 µL of 1 M LiClO4, precipitated with 4% LiClO4 in acetone, and dissolved in 40 µL of water and 20 µL of 5x loading solution. The target oligonucleotide was purified using denaturing PAGE as described above, dissolved in 10 µL of water and subjected to MALDI-TOF analysis on a REFLEX III instrument (Bruker Daltonics, Germany).
6
Synthesis and Characterization of Oligoribonucleotides
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Oligoribonucleotides (strand 1: 5′-AAAUCUGAAAGCCUGACACUUA-3′ and strand 2: 5′-GUGUCAGGCUUUCAGAUUUUUU-3′) were synthesized on an automatic ASM-800 DNA/RNA synthesizer (Biosset) using ribo-β-cyanoethyl phosphoramidites (Glen Research). After standard deprotection, oligoribonucleotides were purified using denaturing polyacrylamide gel electrophoresis (PAGE) and were then isolated as sodium salts. Oligoribonucleotides were characterized by MALDI-TOF mass spectra on REFLEX III (Bruker Daltonics, Germany). isRNAs were annealed at a concentration of 50 μM in a buffer containing 30 mM HEPES-KOH (pH 7.4), 100 mM sodium acetate, and 2 mM magnesium acetate.
High purity polyinosinic-polycytidylic acid (poly(I:C)) (less than 1% free nucleotides) was purchased from Sigma-Aldrich.
High purity polyinosinic-polycytidylic acid (poly(I:C)) (less than 1% free nucleotides) was purchased from Sigma-Aldrich.
7
Protein Identification via Mass Spectrometry
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Coomassie-stained protein bands were excised and prepared as described [38 (link)]. Briefly, excised and chopped bands were washed, destained and digested by trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega, Mannheim, Germany) overnight. The ZipTip (C18, Millipore Corporation, Billerica, MA, US) eluates of the obtained tryptic fragments were mixed 1:1 (v/v) with a 12 mg/mL α-cyano-4-hydroxycinnamic acid matrix (Bruker Daltonics, Bremen, Germany), dissolved in a 2:1 (v/v) mixture of 100% acetonitrile/0.3% TFA, and spotted on the target. Tryptic mass fingerprinting was performed as described previously [39 (link)] using a Reflex III (Bruker Daltonics, Bremen, Germany) in reflector mode, while applying an acceleration voltage of 20 kV. External mass calibration was performed with peptide standard II (Bruker Daltonics, Bremen, Germany). Mascot Peptide Mass Fingerprint (http://matrixscience.com ) and NCBInr database were used to identify digested fragments. For database search the following filters were applied: taxonomy on other green plants, peptide tolerance of ± 0.3 Da and up to one allowed missed cleavage. Variable modifications were the oxidation of methionine residues and N-terminal acetylation.
8
MALDI-TOF MS Analysis of Condensed Tannins
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The MALDI-TOF MS analyses were performed on a Bruker Reflex III instrument (Germany). The measurements were carried out using the conditions reported in the previous work [29] . Reflectron modes coupled with linear modes were further applied to show the different DP distribution of condensed tannins.
9
MALDI-TOF-MS for Peptide Analysis
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MALDI-TOF-MS was performed according to the method described by Zhang et al.16 (link). The MALDI-TOF-MS spectra were recorded on a Bruker Reflex III instrument (Germany). The irradiation source was a pulsed nitrogen laser with a wavelength of 337 nm, and the duration of the laser pulse was 3 ns. In the positive reflection mode, an accelerating voltage of 20.0 kV and a reflectron voltage of 23.0 kV were used. Spectra of PAs were obtained from a sum of 100–150 shots and calibrated using angiotensin II (1,046.5 MW), bombesin (1,619.8 MW), ACTHclip18–39 (2,465.2 MW), and somatostatin 28 (3,147.47 MW) as external standards. 2,5- dihydroxybenzoic acid (DHB, 10 mg/mL aqueous solution) was used as the matrix. The sample solutions (7.5 mg/mL aqueous) were mixed with the matrix solution at a volumetric ratio of 1:3. The mixture (1 μL) was applied to the steel target. Amberlite IRP-64 cation-exchange resin (Sigma-Aldrich), equilibrated in deionized water, was used to deionize the analyte/matrix solution three times. Cesium chloride (1 mg/mL) was mixed with the analyte/matrix solution at 1:3 volumetric ratio to promote the formation of a single type of ion adduct ([M + Cs]+)68 69 .
10
NMR and Mass Spectrometry Analysis
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1H NMR and 13C NMR spectra were recorded at room temperature on a Bruker AMX400. High temperature NMR spectroscopy was conducted on a Bruker AMX500. All NMR measurements were done in CDCl3, C2D2Cl4, or CD3OD/CDCl3 mixture, respectively, with the solvent residual peak as an internal reference [CHCl3: δ = 7.24 p.p.m. (1H) and 77.0 p.p.m. (13C), C2HDCl4: δ = 6.00 p.p.m. (1H), CHD2OD: δ = 3.30 p.p.m. (1H)]. Mass spectrometry (MALDI-TOF) was performed on Bruker Reflex III. The elemental composition was determined with an Elementar Vario EL system. The synthesis of the precursors can be found in Supplementary Information.
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1H NMR and 13C NMR spectra were recorded at room temperature on a Bruker AMX400. High temperature NMR spectroscopy was conducted on a Bruker AMX500. All NMR measurements were done in CDCl3, C2D2Cl4, or CD3OD/CDCl3 mixture, respectively, with the solvent residual peak as an internal reference [CHCl3: δ = 7.24 p.p.m. (1H) and 77.0 p.p.m. (13C), C2HDCl4: δ = 6.00 p.p.m. (1H), CHD2OD: δ = 3.30 p.p.m. (1H)]. Mass spectrometry (MALDI-TOF) was performed on Bruker Reflex III. The elemental composition was determined with an Elementar Vario EL system. The synthesis of the precursors can be found in Supplementary Information.
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