Structural analysis was performed by HSQC NMR spectroscopy. For analysis, 60 mg of lignin was dissolved in 0.7 mL of d6-acetone. A few drops of D2O were added to ensure complete solubility of the lignin. The NMR analysis was performed on a Bruker Ascend™ Neo 600 using the following parameters: (F2 = 11 to −1 ppm), (F1 = 160 to −10 ppm), nt = 4, ni = 512, d1 = 1.5, CNST [2 (link)] = 145, and pulse sequence hsqcetgpsi2. Analysis was performed with MestReNova. The obtained values for the detectable linking motifs (β-O-4, β′-O-4, β-β, and β-5) were divided by a factor of 1.3 as the HSQC measurements overestimate these values as was shown in previous work [46 (link)].
Neo 600
Manufactured by Bruker
The NEO 600 is a high-performance nuclear magnetic resonance (NMR) spectrometer designed for analytical and research applications. It provides a flexible and reliable platform for conducting advanced NMR experiments. The NEO 600 features a 14.1 Tesla superconducting magnet and offers a wide range of capabilities for various fields of study.
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Lab products found in correlation
Topspin 4, by Bruker (1 mentions)
Ethyl methacrylate, by Merck Group (1 mentions)
Ethyl methacrylate, by Thermo Fisher Scientific (1 mentions)
1515 hplc instrument, by Waters Corporation (1 mentions)
2 2 azobisisobutyronitrile, by Merck Group (1 mentions)
Mr 400, by Agilent Technologies (1 mentions)
Methyl 3 mercaptopropionate, by Thermo Fisher Scientific (1 mentions)
Trifluoroacetic acid tfa and solvents, by Thermo Fisher Scientific (1 mentions)
Synergy lx, by Agilent Technologies (1 mentions)
Dsc q20 calorimeter, by TA Instruments (1 mentions)
6 protocols using neo 600
1
Quantitative Lignin Analysis by HSQC NMR
2
Structural Characterization of RCAN1 by NMR
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NMR data were collected on either Bruker NEO 600- and 800-MHz spectrometers or a Bruker Avance III HD 850-MHz spectrometer equipped with TCI HCN z-gradient cryoprobes at 298 K. NMR measurements of RCAN1 were recorded using (1H,15N)-, (2H,15N)-, (1H,15N,13C)-, or selectively γ/δ-[13CH3, 12CD3]-Val/Leu, δ-[13CH3]-Ile, [U]-2H,15N-labeled protein at a final concentration of 0.6 mM in NMR buffer and 90% H2O/10% D2O. The sequence-specific backbone assignments of RCAN1 and variants, as well as CN-bound RCAN1 were achieved using 3D triple-resonance experiments including 2D [1H,15N] HSQC/TROSY, 3D HNCA, 3D HN(CO)CA, 3D HN(CO)CACB, and 3D HNCACB. All NMR data were processed using TopSpin 4.05 (Bruker BioSpin) and analyzed using CARA (http://cara.nmr.ch ) and/or CcpNMR (40 ). 2D [1H,13C]-HMQC, 3D 13C-ILV-methyl-methyl resolved [1H,1H] NOESY, 13C-methyl-ILV-15N resolved [1H,1H] NOESY, and 15N-resolved [1H,1H] NOESY spectra were recorded with a mixing time (TM) of 120 ms using the selectively γ/δ-[13CH3, 12CD3]-Val/Leu, δ-[13CH3]-Ile, [U]-2H,15N-labeled RCAN1 and [U]-2H-labeled CNA complex. The NOE data were also used to assign the chemical shifts of γ/δ-CH3 of Val/Leu, δ-[CH3]-Ile.
3
Synthesis and Characterization of Functionalized Monomers
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tert-Butyl-3-hydroxypropanoate, methyl 3-mercaptopropionate, and ethyl methacrylate were purchased from Acros Organics. 2,2′-Azobis-(isobutyronitrile) (AIBN) and ethyl methacrylate were purchased from Sigma-Aldrich Co. Trifluoroacetic acid (TFA) and solvents were purchased from Thermo Fisher Scientific, Inc. The chemicals were used without further purification, with the exception of methacryloyl chloride which was distilled before use and AIBN which was recrystallized from hot methanol. Boc-amino ethyl methacrylate was synthesized according to the previous reported procedure.61 (link)1H NMR was performed using a Varian MR400 (400 MHz), 13C NMR was performed using a Bruker NEO600 (600 MHz) and the data was analyzed using VNMRJ 3.2 and MestReNova. Gel permeation chromatography (GPC) analysis was performed using a Waters 1515 HPLC instrument equipped with Waters Styragel (7.8 × 300 mm) HR 0.5, HR 1, and HR 4 columns in sequence and detected by a differential refractometer (RI). Mueller Hinton broth (MHB, BD and Company) and phosphate buffered saline (PBS, pH = 7.4, Gibco) were prepared according to manufacturer instructions and sterilized prior to use. Human red blood cells (RBCs) (leukocytes reduced adenine saline added) were obtained from the American Red Cross Blood Services Southeastern Michigan Region and used prior to the out date indicated on each unit.
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tert-Butyl-3-hydroxypropanoate, methyl 3-mercaptopropionate, and ethyl methacrylate were purchased from Acros Organics. 2,2′-Azobis-(isobutyronitrile) (AIBN) and ethyl methacrylate were purchased from Sigma-Aldrich Co. Trifluoroacetic acid (TFA) and solvents were purchased from Thermo Fisher Scientific, Inc. The chemicals were used without further purification, with the exception of methacryloyl chloride which was distilled before use and AIBN which was recrystallized from hot methanol. Boc-amino ethyl methacrylate was synthesized according to the previous reported procedure.61 (link)1H NMR was performed using a Varian MR400 (400 MHz), 13C NMR was performed using a Bruker NEO600 (600 MHz) and the data was analyzed using VNMRJ 3.2 and MestReNova. Gel permeation chromatography (GPC) analysis was performed using a Waters 1515 HPLC instrument equipped with Waters Styragel (7.8 × 300 mm) HR 0.5, HR 1, and HR 4 columns in sequence and detected by a differential refractometer (RI). Mueller Hinton broth (MHB, BD and Company) and phosphate buffered saline (PBS, pH = 7.4, Gibco) were prepared according to manufacturer instructions and sterilized prior to use. Human red blood cells (RBCs) (leukocytes reduced adenine saline added) were obtained from the American Red Cross Blood Services Southeastern Michigan Region and used prior to the out date indicated on each unit.
4
600 MHz NOE Spectroscopy in CDCl3
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Selective NOE 1H spectra were recorded on a Bruker NEO 600 instrument (prodigy BBO probe) at 600.18 MHz at the lower measured temperature (0.1 M in 0.6 mL CDCl3). 1H NMR spectra were acquired using the selnogp pulse program, an 80 ms selective Gaussian pulse, a mixing time of 1 s, a spectral width of 10 kHz, an acquisition time of 4.6 s, and 256 scans, zerofilled to 64 k datapoints (0.15 Hz per point) and processed with EM (LB=1) apodization.
5
Polymer Tensile Properties and Characterization
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An ASTM D412 Type D cutting die was purchased
from Universal Grip Co. Tensile measurements were performed using
an Instron 5900 series Universal Mechanical Tester. Young’s
modulus was taken at 10% strain. UV–vis measurements were performed
using a BioTek Synergy LX multi-mode plate reader. 1H NMR
spectra were obtained using a Bruker NEO 600. IR spectra were obtained
using a Nicolet 6700 FT-IR spectrometer. Dynamic scanning calorimetry
was measured on a TA instruments DSC Q20 calorimeter with a heating
ramp of 10 °C·min–1 under a N2 atmosphere.
from Universal Grip Co. Tensile measurements were performed using
an Instron 5900 series Universal Mechanical Tester. Young’s
modulus was taken at 10% strain. UV–vis measurements were performed
using a BioTek Synergy LX multi-mode plate reader. 1H NMR
spectra were obtained using a Bruker NEO 600. IR spectra were obtained
using a Nicolet 6700 FT-IR spectrometer. Dynamic scanning calorimetry
was measured on a TA instruments DSC Q20 calorimeter with a heating
ramp of 10 °C·min–1 under a N2 atmosphere.
6
NMR Characterization of MAX Protein
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TROSY and HSQC spectra were recorded using a Bruker NEO 600-, 700-, or 850-MHz spectrometer equipped with cryogenically cooled probe heads optimized for 1H detection. Spectra were recorded in the States-TPPI/PFG mode for quadrature detection with carrier frequencies for 1HN and 15N of 4.73 and 120.0 ppm, respectively. The samples contained 1 μM, 0.1 mM, or 0.3 mM MAX; 25 mM MES; 100 mM ArgHCl; and 25 mM NaCl (pH 5.5) in a 90% H2O/10% D2O mixture.
All NMR spectra were processed and analyzed using TOPSPIN 4.0.7 and MATLAB R2019a. NMRPipe and SPARKY were used to process and analyze the recorded TROSY and HSQC data (54 , 55 (link)). A squared and 60° phase-shifted sine bell window function was applied in all dimensions for apodization. Time-domain data were zero-filled to twice the dataset size, before Fourier transformation.
In addition, we considered the possibility that a shift in pH could influence MAX’s configuration. Therefore, we performed a TROSY experiment on MAX in a 30 times diluted buffer (fig. S5). Despite the significative variation of pH (from 5.5 to 7.15) in the lowly concentrated buffer, the detected spectrum does not show any similarities with the spectra obtained from a lowly concentrated protein sample (fig. S4C).
All NMR spectra were processed and analyzed using TOPSPIN 4.0.7 and MATLAB R2019a. NMRPipe and SPARKY were used to process and analyze the recorded TROSY and HSQC data (54 , 55 (link)). A squared and 60° phase-shifted sine bell window function was applied in all dimensions for apodization. Time-domain data were zero-filled to twice the dataset size, before Fourier transformation.
In addition, we considered the possibility that a shift in pH could influence MAX’s configuration. Therefore, we performed a TROSY experiment on MAX in a 30 times diluted buffer (fig. S5). Despite the significative variation of pH (from 5.5 to 7.15) in the lowly concentrated buffer, the detected spectrum does not show any similarities with the spectra obtained from a lowly concentrated protein sample (fig. S4C).
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