Overnight cultures of E coli cells transformed with GES-19, GES-26, or GES-19/GES-26 in the IPTG-inducible pBA169 expression plasmid were diluted 1:100 in 50 mL LB medium containing 25 µg/mL chloramphenicol (to maintain the plasmid). The diluted cultures were incubated with shaking at 37oC to OD600 of 0.8. The IPTG was added to a final concentration of 0.5 mM, and cells were incubated with shaking at 20oC for another 20 hours to induce the expression of GES proteins. Cells were then pelleted by centrifugation at 6000 rpm for 20 minutes, and the cell pellet was resuspended in 2 mL B-PER (Thermo Fisher) containing 100 µg/mL lysozyme and 20 µg/mL DnaseI and incubated at room temperature for 15 minutes. Samples were then centrifuged again at 13000 rpm for 5 minutes to collect cell lysate in the supernatant. To ensure the expression of GES enzymes, 50 µM nitrocefin diluted in 50 mM HEPES (pH7.4) was incubated with 1 µL cell lysate, and absorbance at 482 nm (A482) was monitored on Beckman DU800 spectrometer using a 1-cm cuvette. To determine hydrolysis of ceftazidime by the GES enzymes, 50 µM ceftazidime diluted in 50 mM HEPES (pH7.4) was incubated with 1 µL cell lysate, and absorbance at 260 nm (A260) was monitored on Beckman DU800 spectrometer using a 1-cm cuvette.
Du 800 spectrometer
Manufactured by Beckman Coulter
Sourced in United States, Japan
The DU 800 spectrometer is a UV-Vis spectrophotometer designed for accurate and reliable absorbance measurements. It features a dual-beam optical system, a high-intensity xenon lamp, and a linear CCD array detector. The DU 800 is capable of performing a wide range of spectroscopic analyses, including quantitative determinations, kinetic studies, and DNA/protein analysis.
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Lab products found in correlation
Ftir 8400s spectrophotometer, by Shimadzu (3 mentions)
Silica gel g precoated plates, by Qingdao Haiyang Chemical (3 mentions)
241 polarimeter, by Beckman Coulter (2 mentions)
Sephadex lh 20, by GE Healthcare (2 mentions)
241 polarimeter, by Jasco (1 mentions)
Tof esi ms waters synapt g2, by Waters Corporation (1 mentions)
Rp 18, by YMC (1 mentions)
J 815 spectropolarimeter, by Jasco (1 mentions)
Gf254, by Qingdao Marine Chemical (1 mentions)
Lh 20, by Merck Group (1 mentions)
Api qstar pulsar mass spectrometer, by Bruker (1 mentions)
Av 500 spectrometer, by Bruker (1 mentions)
J 815 spectrometer, by Jasco (1 mentions)
Silica gel, by Qingdao Haiyang Chemical (1 mentions)
Amicon ultra centrifugal filter device, by Merck Group (1 mentions)
Cdpx 300, by Bruker (1 mentions)
Av 360, by Bruker (1 mentions)
Dpx 300, by Bruker (1 mentions)
Drx 400, by Bruker (1 mentions)
Directdrive nmr system, by Agilent Technologies (1 mentions)
12 protocols using du 800 spectrometer
1
Expression and Characterization of GES Enzymes
2
Analytical Techniques for Natural Product Characterization
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Optical rotations were obtained on a PerkinElmer 241 Polarimeter (Waltham, MA, USA), and CD measurements were performed with a JASCO J-815 Spectropolarimeter. UV data were measured using a Beckman Coulter DU 800 spectrometer (Tokyo, Japan). IR spectra were recorded using a Shimadzu FTIR-8400S spectrophotometer (Tokyo, Japan). NMR spectra were acquired with a Bruker 500 (Munich, Germany) instruments operating at 500 (1H) and 125 (13C) MHz using the solvent signals (CD3OD: δH 3.31/δC 49.0 ppm) as references. The HRESIMS experiments were carried out on a TOF-ESI-MS (Waters Synapt G2, USA) equipment. HPLC for purifications were performed on a Waters 2489 Semiprep-HPLC System using an ODS column (RP-18, 250 × 10 mm, YMC Pack, 5 μm; detector: UV; Kyoto, Japan). Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) and silica gel (80–100, 100–200, and 200–300 mesh; Qingdao Marine Chemical Plant, Qingdao, China) were used for column chromatography, and thin layer chromatography (TLC) was carried out with glass precoated silica gel GF254 (0.20–0.25 mm; Qingdao Marine Chemical Plant, Qingdao, China).
3
Comprehensive Nanomaterial Characterization Protocol
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For characterization of material, the morphology was assessed using a high-resolution JEOL JEM-2100Plus TEM (Hitachi Scientific Instruments, Japan). The FT-IR spectra were recorded with a Thermo-Nicolet Nexus 6700 FT-IR spectrometer (USA). The UV–Vis spectra were measured with a DU800 spectrometer (Beckman Coulter Inc., USA). The size and zeta potential of nanomaterial were measured with a Zetasizer Nano analyzer (Malvern Nano Series, UK). The chemical states of the elements were measured with an X-ray photoelectron spectroscopy (XPS) (ULVAC-PHI Inc., Japan).
A fluorescence assay was illustrated to characterize the extent of membrane fusion. Briefly, the DiI labeled RBCM and DiO labeled 231 M were mixed with equal weight. The fluorescence of the hybrid membrane was imaged under confocal laser scanning microscope (CLSM).
SDS-PAGE was employed to characterize the integrity of membrane proteins. Briefly, RBCM, 231 M, HM, and CPCCM were lysed with membrane protein extraction reagent B containing 1 mM PMSF. The protein concentration of all samples was detected using the BCA Kit, followed by proteins (20 μg) denature at 95 °C for 10 min. The proteins were separated in the 12% SDS-PAGE gel and stained using Coomassie Brilliant Blue solution.
A fluorescence assay was illustrated to characterize the extent of membrane fusion. Briefly, the DiI labeled RBCM and DiO labeled 231 M were mixed with equal weight. The fluorescence of the hybrid membrane was imaged under confocal laser scanning microscope (CLSM).
SDS-PAGE was employed to characterize the integrity of membrane proteins. Briefly, RBCM, 231 M, HM, and CPCCM were lysed with membrane protein extraction reagent B containing 1 mM PMSF. The protein concentration of all samples was detected using the BCA Kit, followed by proteins (20 μg) denature at 95 °C for 10 min. The proteins were separated in the 12% SDS-PAGE gel and stained using Coomassie Brilliant Blue solution.
4
Characterization of Novel Compounds
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The IR spectra (KBr pellets) were run on a 380 FT-IR instrument from Nicolet (Thermo, Pittsburgh, PA, USA). The HRMS were recorded with an API QSTAR Pulsar mass spectrometer (Bruker, Bremen, Germany). The UV spectra were obtained from a DU-800 spectrometer (Beckman, Brea, CA, USA). Optical rotations were measured on an Autopol III polarimeter (Rudolph, Hackettstown, NJ, USA). CD spectra were recorded with a J-815 spectrometer (JASCO, Tokyo, Japan). The NMR spectra were recorded on an AV-500 spectrometer (500 MHz for 1H-NMR and 125 MHz for 13C-NMR; Bruker), using the solvent residue signal as the internal standard. Column chromatography was performed with ODS gel (20–45 mm, Fuji Silysia Chemical Co. Ltd., Durham, NC, USA), Sephadex LH-20 (Merck, Darmstadt, Germany) and silica gel (60–80, 200–300 mesh, Qingdao Haiyang Chemical Co. Ltd., Qingdao, China). TLC was carried out on silica gel G precoated plates (Qingdao Haiyang Chemical Co. Ltd.), and spots were detected by spraying with 5% H2SO4 in EtOH followed by heating.
5
Spectroscopic Characterization of Compounds
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Optical rotations were measured on a Perkin-Elmer 241 polarimeter, and UV data were recorded on Beckman Coulter DU 800 spectrometer. IR data were recorded using a Shimadzu FTIR-8400S spectrophotometer. 1H and 13C NMR data were acquired with a Bruker 600 spectrometer using solvent signals (CDCl3; δH 7.26/δC 77.6, CD3OD; δH 3.31/δC 49.9, DMSO-d6;δH 2.49/δC 39.5) as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HRESIMS data were acquired using a LTQ Orbitrap XL mass spectrometer.
6
Comprehensive Animal Plasma Biomarker Analysis
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At days 0 (baseline) and 14 of treatment, a blood sample (500 μL) was draw from each experimental animal by retro-orbital plexus puncture using heparinized capillaries which were then poured into microcentrifuge tubes. After centrifugation (2500 rpm, for 5 minutes, at 4°C), plasma samples were submitted to biochemical analysis, using commercially available kits (all from Bioclin/Quibasa, Minas Gerais, Brazil), including the quantitation of kinetic creatinine (KC, cat. #K067), creatine kinase MB (CK-MB, cat.# K069), total creatine kinase (total CK, cat.# K010), urea (U, cat.# K056), lactic dehydrogenase (LDH cat.# K014), aspartate aminotransferase (ALT, cat.# K048), alanine aminotransferase (ALT, cat.# K049), and alkaline phosphatase (ALP, cat.# K021). All samples were read on a Beckman Coulter DU 800 spectrometer (Beckman Instruments, California, USA).
7
Monitoring Alkaline Transition in Cytochrome c
8
Spectroscopic Determination of SN-38 Binding
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An excess amount of SN-38 was added to PBS buffer (pH 7.4). The SN-38/PBS suspension was pre-incubated at 37°C for 30 min, and then mixed with an L-PGDS solution. This solution was then stirred at 37°C for 6 h and thereafter concentrated by using an Amicon Ultra Centrifugal Filter Device (Millipore Corporation, Bedford, MA). The absorption spectrum of the filtrate was obtained by use of a 1.0 cm-light-path quartz cuvette and DU800 spectrometer (Beckman Coulter, Pasadena, CA). The concentrations of SN-38 were determined spectroscopically based on the molar absorption coefficient of ε380 in DMSO for SN-38 = 20,985 M-1 cm-1.
9
Synthesis and Characterization of Reagent 1
10
Comprehensive Characterization of Organic Compounds
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Melting point determination was performed on a PTP-M apparatus (Khimlabpribor, Klin, Russia). The infrared (IR) spectrum was recorded in CHCl3 on a Specord 75IR spectrometer (Analytik Jena, Jena, Germany) and the ultraviolet (UV) spectrum was recorded in EtOH on a Beckman Coulter DU 800 spectrometer (Beckman Coulter Inc., Brea, CA, USA). 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on a Varian DirectDrive NMR System (Varian, Palo Aho, CA, USA) in CDCl3 at 700 and/or 175.8 MHz. CDCl3 was used as an internal standard. Mass spectrometry with electrospray ionization MS (ESI) was performed using LC-MS high-resolution spectrometer MaXis (ESI-QTOF) (Bruker Corporation, Billerica, MA, USA), with direct injections of sample solutions into the ionization chamber. X-ray diffraction was measured on Kappa Apex II device (Bruker Corporation, Billerica, MA, USA) by use of CuKα radiation (λ = 1.54178 Å). Intensity of reflexes was integrated and scaled by means of the Saint and Sadabs programs from an Apex II package (Bruker AXS Inc., Madison, WI, USA). All subsequent procedures for the decision and specification of the compound structure were held in the software package of Olex2 (https://www.olexsys.org/olex2/ , accessed on 1 January 2020) [44 (link)].
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