In-gel fluorescence scanning was performed using a Typhoon 9410 variable mode imager (excitation 532 nm, emission 580 nm). Isothermal titration calorimetry measurements were performed on a MicroCal iTC200 titration calorimeter (Malvern Instruments, United Kingdom). Peptides were purified on a preparative HPLC system with Waters (Milford, MA) 2535 Quaternary Gradient Module, Waters 515 HPLC pump, Waters SFO System Fluidics Organizer, and Waters 2767 Sample Manager. Enzymatic reactions were monitored by an LC-MS system with Waters 1525 Binary HPLC Pump, Waters 2998 Photodiode Array Detector, and Waters 3100 Mass Detector. Detection of O-Cr-ADPR was carried out by Agilent (Santa Clara, CA) 1260 Infinity HPLC system connected to a Thermo Fisher Scientific LCQ DecaXP MS detector.
2535 quaternary gradient module
Manufactured by Waters Corporation
Sourced in United States
The 2535 Quaternary Gradient Module is a component of a liquid chromatography system designed to deliver precise and accurate gradient elution. It is capable of generating linear, stepped, or curved gradients using up to four independent solvents. The module's core function is to precisely control the flow and composition of the mobile phase during the chromatographic separation process.
Automatically generated - may contain errors
Lab products found in correlation
Sfo system fluidics organizer, by Waters Corporation (5 mentions)
515 hplc pump, by Waters Corporation (4 mentions)
Waters 515 hplc pump, by Waters Corporation (2 mentions)
Waters 2767 sample manager, by Waters Corporation (2 mentions)
2767 sample manager, by Waters Corporation (2 mentions)
Waters 3100 mass detector, by Waters Corporation (1 mentions)
1260 infinity hplc system, by Thermo Fisher Scientific (1 mentions)
Lcq decaxp ms detector, by Thermo Fisher Scientific (1 mentions)
1525 binary hplc pump, by Waters Corporation (1 mentions)
Waters 2998 photodiode array detector, by Waters Corporation (1 mentions)
Typhoon 9410 variable mode imager, by GE Healthcare (1 mentions)
Enf 260c fe hand hang uv lamp, by Spectroline (1 mentions)
Puriflash 450, by Interchim (1 mentions)
Flash c18 cartridge, by YMC (1 mentions)
Lambda 365 uv vis spectrophotometer, by PerkinElmer (1 mentions)
Avance 400 mhz spectrometer, by Bruker (1 mentions)
Lcq deca xp, by Thermo Fisher Scientific (1 mentions)
Agilent 1260 infinity manual injector, by Agilent Technologies (1 mentions)
Agilent 1260 infinity quaternary pump vl, by Agilent Technologies (1 mentions)
300 or 400 mhz spectrometer, by Bruker (1 mentions)
7 protocols using 2535 quaternary gradient module
1
Biophysical Characterization of Biomolecules
2
Analytical Characterization of Peptides
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1H NMR (300 MHz or 400 MHz), 13C NMR (75 MHz, 100 MHz) were conducted on a Bruker spectrometer at 25 °C and were calibrated using residual undeuterated solvent as an internal reference. Chemical shifts were reported in ppm and coupling constants (J) were quoted to the nearest 0.1 Hz. High resolution mass spectrometry (HRMS) was recorded using a Bruker maXis II High Resolution QTOF. Peptides were analyzed by LC-MS with an Agilent 1260 Infinity HPLC system connected to a Thermo Finnigan LCQ DecaXP MS detector. Peptides were further purified by a preparative HPLC system with Waters 2535 Quaternary Gradient Module, Waters 515 HPLC pump, Waters SFO System Fluidics Organizer and Waters 2767 Sample Manager.
Photo-cross-linking were performed with ENF-260C/FE hand-hang UV lamp (Spectroline). In-gel fluorescence scanning was performed using a Typhoon 9410 variable mode imager from GE Healthcare Life Sciences (excitation 532 nm, emission 580 nm). All images were processed by ImageJ software (National Institutes of Health), and contrast was adjusted appropriately.
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1H NMR (300 MHz or 400 MHz), 13C NMR (75 MHz, 100 MHz) were conducted on a Bruker spectrometer at 25 °C and were calibrated using residual undeuterated solvent as an internal reference. Chemical shifts were reported in ppm and coupling constants (J) were quoted to the nearest 0.1 Hz. High resolution mass spectrometry (HRMS) was recorded using a Bruker maXis II High Resolution QTOF. Peptides were analyzed by LC-MS with an Agilent 1260 Infinity HPLC system connected to a Thermo Finnigan LCQ DecaXP MS detector. Peptides were further purified by a preparative HPLC system with Waters 2535 Quaternary Gradient Module, Waters 515 HPLC pump, Waters SFO System Fluidics Organizer and Waters 2767 Sample Manager.
Photo-cross-linking were performed with ENF-260C/FE hand-hang UV lamp (Spectroline). In-gel fluorescence scanning was performed using a Typhoon 9410 variable mode imager from GE Healthcare Life Sciences (excitation 532 nm, emission 580 nm). All images were processed by ImageJ software (National Institutes of Health), and contrast was adjusted appropriately.
3
Fmoc-Lys(tBu-Succinyl)-OH Peptide Synthesis
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The unnatural amino acid Fmoc-Lys(tBu–Succinyl)-OH used for peptide synthesis was synthesized as reported previously (20 (link)). The peptide used in this research was synthesized on 2-chlorotrityl chloride resin following standard Fmoc-based solid-phase peptide synthesis protocol. After the coupling of all amino acids, the removal of protecting groups and cleavage of peptides from the resin were done by incubating the resin with cleavage cocktail containing 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane, 1.5% water and 1% thioanisole for 2 h. Peptides were purified on a preparative high performance liquid chromatography (HPLC) system (Waters 2535 Quaternary Gradient Module, Waters 515 HPLC pump) with a Vydac C18 column (22 mm × 250 mm, 10 μm, Garce). The purity (>95%) and identity of peptide was confirmed by LC–MS.
4
Purification of Synthesized Proteins
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All the buffers were prepared with ddH2O using the standard methods. The synthesized proteins were purified on a preparative high performance liquid chromatography (HPLC) system with a Waters 2535 Quaternary Gradient Module, a Waters 515 HPLC pump, a Waters SFO system Fluidics Organizer and a Waters 2767 Sample Manager. The synthesis of compound 1 was performed following a previously reported method.14 (link)
5
Spectroscopic and Chromatographic Analysis of Compounds
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Optical rotation values were determined on a JASCO P-1010 polarimeter (Jasco, Tokyo, Japan). UV spectra were recorded on a PerkinElmer Lambda 365 UV-Vis spectrophotometer (PerkinElmer, Hopkinton, MA, USA). High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) data were measured on a Waters ACQUITY UPLC H-Class Q-TOF LC-MS spectrometer (Waters, Milford, MA, USA). High-performance liquid chromatography (HPLC) analysis was carried out on an ACQUITY UPLC H-Class System (quaternary solvent manager, sample manager, PDA detector, and ELS detector) using a YMC ODS (4.6 × 250 mm, 5 µm, 1 mL/min) column. MPLC was performed on a PuriFlash450 (Interchim, Los Angeles, CA, USA) with a Flash C18 cartridge (50 µm, 40 g, YMC, Kyoto, Japan). Semipreparative HPLC was performed on a Waters 2535 Quaternary gradient module with a FlexInject, 2489 UV–VIS detector and Fraction Collector Ⅲ (Waters, Milford, MA, USA). The NMR spectra were recorded on a Bruker Avance 400 MHz spectrometer using tetramethylsilane as the internal standard (Bruker, Ettlingen, Germany). Thin-layer chromatography (TLC) analyses were performed on glass precoated with silica gel GF254 glass plates. All reagents for the analysis were purchased from Xilong Scientific Co., Ltd. (Guangdong, China).
6
Purification and Characterization of Peptides
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All NMR spectra were recorded on a Bruker 300 or 400 MHz spectrometer. The chemical shifts were reported in ppm, and J values were reported in Hz. Peptides were purified on a Grace VYDACÒ 218TP152025 C18 column connected to a preparative-HPLC system with Waters 2535 Quaternary Gradient Module, Waters 515 HPLC pump, Waters SFO system Fluidics Organizer and Waters 2767 Sample Manager. Analytical HPLC trace after purification was obtained using a Grace VYDACÒ 218TP C18 5m column connected to an HPLC system with Agilent 1260 Infinity Quaternary Pump VL, Agilent 1260 Infinity Manual Injector and Agilent 1260 Infinity Variable Wavelength Detector. The outlet of the above HPLC system was connected to Thermo Finnigan LCQ Deca XP to obtain MS spectrums of purified peptides.
Starting materials for organic synthesis were purchased from common commercial suppliers including Sigma-Aldrich, TCI and Alfa and used without further purification. All reactions were monitored by TLC Silica gel 60 F254 from Merck. Flash column chromatography was performed with silica gel purchased from Grace (40-63 micron). All Fmoc-protected amino acids for and coupling reagents for solid phase peptide synthesis were purchased from GL Biochem (Shanghai, China).
Starting materials for organic synthesis were purchased from common commercial suppliers including Sigma-Aldrich, TCI and Alfa and used without further purification. All reactions were monitored by TLC Silica gel 60 F254 from Merck. Flash column chromatography was performed with silica gel purchased from Grace (40-63 micron). All Fmoc-protected amino acids for and coupling reagents for solid phase peptide synthesis were purchased from GL Biochem (Shanghai, China).
7
Fmoc-based Solid-Phase Peptide Synthesis
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All amino acids and coupling reagents were purchased from GL Biochem (Shanghai, China). All the peptides used in this research were synthesized on Rink-Amide MBHA resin or 2-Chlorotrityl chloride resin following standard Fmoc-based solid-phase peptide synthesis protocol. After the coupling of all amino acids, the removal of protecting groups and cleavage of peptides from the resin were done by incubating the resin with cleavage cocktail containing 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane, 1.5% water and 1% thioanisole for 2 h. Peptides were purified on a preparative high performance liquid chromatography (HPLC) system (Waters 2535 Quaternary Gradient Module, Waters 515 HPLC pump, Waters SFO system Fluidics Organizer and Waters 2767 Sample Manager) with a Vydac C18 column (22 mm X 250 mm, 10 mm, Garce). Mobile phase used were water with 0.1% TFA and 90% acetonitrile in water with 0.1% TFA. The purity (> 95%) and identity of peptides were confirmed by LC-MS, for details see Supplemental Information.
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