Full length DVU0636, DVU2067, and DVU0408 with a C-terminal V5 epitope and 6x His tag were purified from E. coli. Purified protein (1.6–1.8 μM) was mixed with 0.5 mM GTP in 50 mM Tris HCl pH 8.0, 100 mM NaCl, and either 2 mM MgCl2 or 2 mM MnCl2 in a total volume of 100 μl. The reactions were incubated at 30°C for 24 h. The proteins were denatured by heating at 95°C for 5 min. The samples were centrifuged at 15,000 × g for 10 min, and the supernatant was filtered through a 10 K molecular weight cutoff centrifugal filter prior to HPLC analysis. Samples (3 μl) were injected into an Inertsil ODS-3 column (3 μ, 250 × 2.1 mm; GL Sciences, Torrance, CA) equipped with a guard column (Inertsil ODS-3, 3 μ, 50 × 2.1 mm; GL Sciences) on an HPLC system (Agilent Technologies, Santa Clara, CA). Buffer A: 100 mM Potassium phosphate buffer pH 6.0; Buffer B = 100% (vol/vol) methanol. The samples were run for 25 min at a flow rate of 0.2 ml/min, with the following gradient: 0 min – 2% (vol/vol) B; 2 min – 2% B; 14 min – 30% B; 17 min – 30% B; 18 min – 2% B; 25 min – 2%B.
Inertsil ods 3
Manufactured by GL Sciences
Sourced in Japan, United States
Inertsil ODS-3 is a reversed-phase high-performance liquid chromatography (HPLC) column. It is designed for the separation and analysis of a wide range of organic compounds. The column features a silica-based stationary phase with octadecylsilane (ODS) bonding, providing a hydrophobic surface for the retention of non-polar and moderately polar analytes.
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Lab products found in correlation
Lcms 8040, by Shimadzu (3 mentions)
Api 3000, by AB Sciex (3 mentions)
Hind 3, by Thermo Fisher Scientific (2 mentions)
Ecori, by Thermo Fisher Scientific (2 mentions)
Bamhi, by Thermo Fisher Scientific (2 mentions)
Rnase h buffer, by New England Biolabs (2 mentions)
1290 infinity 2 autosampler and binary pump, by Agilent Technologies (2 mentions)
Sciex 6500 triple quadrupole mass spectrometer, by AB Sciex (2 mentions)
Genejet pcr purification kit, by Thermo Fisher Scientific (2 mentions)
Rnase h, by New England Biolabs (2 mentions)
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Rnase a, by Thermo Fisher Scientific (2 mentions)
Dna degradase plus, by Zymo Research (2 mentions)
Diode array detector, by Agilent Technologies (2 mentions)
Spd m20a, by Shimadzu (2 mentions)
Nexera x2, by Shimadzu (2 mentions)
Agilent 1100 series, by Agilent Technologies (2 mentions)
Polylysine, by Merck Group (2 mentions)
Cellrox deep red reagent, by Thermo Fisher Scientific (2 mentions)
Dapi fluoromount, by Southern Biotech (2 mentions)
67 protocols using inertsil ods 3
1
Purification and HPLC Analysis of GTPase Proteins
2
Qualitative and Quantitative Analysis of Desulpho-GSL
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For qualitative analysis, separation of desulpho-GSL was conducted on a C18 column (250 × 2.1 mm, 5 μm, Inertsil ODS-3; GL Sciences, Tokyo, Japan) using a HPLC system equipped with a diode array detector (Shimadzu, Kyoto, Japan). The elution buffers consisted of solvent A (water) and B (acetonitrile). The flow rate was 0.2 ml/min. The following elution program was applied: 0 min, 99% A/1% B; 18 min, 20% A/80% B; 30 min, 20% A/80% B; 32 min, 99% A/1% B; and 40 min, 99% A/1% B. The UV–Visible detector wavelength was set at 227 nm. For the MS analysis, the eluate was diverted to a mass spectrometer (Hitachi M-800, Tokyo, Japan) equipped with atmospheric pressure chemical ionization (APCI) in a positive mode [M + H+]. The spray voltage was set to 4.5 kV and the capillary temperature was set to 250 °C. The scan of the masses ranged from m/z 100 to m/z 700. For quantitative analysis, desulpho-GSL extracts were separated on a C18 column (250 × 4.6 mm, 5 μm, Inertsil ODS-3; GL Sciences) using a HPLC system equipped with a diode array detector (LC-20A; Shimadzu). The HPLC conditions were the same as described previously, except the flow rate was 1.0 ml/min. GSL content was calculated using sinigrin as an external standard and the response factor of each compound relative to sinigrin.
3
Quantitative Analysis of DNA Composition
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Cells were resuspended in TE buffer containing 62.5 μg/mL proteinase K (Invitrogen, AM2546), 62.5 μg/mL RNase A (Thermo Scientific, EN0531), and 0.5% SDS and incubated overnight at 37°C. Genomic DNA was purified by phenol/chloroform extraction and resuspended in RNase/DNase free water. 5 μg DNA was digested with 5 μL RNase H (NEB, M0297), 3 μL Hind III (Fisher, FD0504), 3 μL EcoRI (Fisher, FD0274), and 3 μL Bam HI (Fisher, FD0054) in RNase H buffer (NEB, M0297) overnight at 37°C. Digested DNA was purified using the GeneJET PCR Purification Kit (Thermo Scientific, K0702). DNA was further digested into single nucleosides using DNA Degradase Plus (Zymo Research, E2021). To quantitate DNA constituent base composition, a fit-for-purpose LC-MS/MS assay was implemented on a 1290 Infinity II Autosampler and Binary Pump (Agilent) and a SCIEX 6500+ triple quadrupole mass spectrometer (SCIEX). Chromatographic separation was conducted on an Inertsil ODS-3 (3 μm × 100 mm 2.1 mm) reverse phase column (GL Sciences) at ambient temperature with a gradient mobile phase of methanol and water with 0.1% formic acid. MRM transitions of all analytes and isotopic internal standards were monitored to construct calibration curves. We were able to quantitate 1 rN per 20,000 bases from 1 mg DNA.
4
Reverse-Phase HPLC Determination of Compounds
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Under the different experimental condition the method was optimized for their different mobile phase solvent ratio, column temperature, flow rate and pH of inorganic mobile phase. Reverse phase HPLC determinations were performed with Agilent technologies (Model-1120, Compact, G4288A-gradient) having gradient low pressure binary pump, consisting of vacuum in built degasser unit, with VWD, equipped with a manual injector system of 50 µl loop. Detection accomplished with a UV detector at 343 nm. Integration and system parameters were controlled by “Ezchrom Elite” compact, software 3.0.1 system. Chromatographic analysis was carried out at 30° on a C-18 column Inertsil® ODS-3 (250×6 mm, 5 µm, GL Sciences Inc. USA) column. The separation was achieved by isocratic elution with a flow rate 2.0 ml/min. The mobile phase was made by inorganic water, and organic (acetonitrile:methanol: 50:50, v/v) with ratio of 75:25, v/v with sodium 1-pentanesulfonate (96 mg/1000 ml of mobile phase). The pH 3.0 of inorganic phase was adjusted with dilute orthophosphoric acid and sodium hydroxide solution.
5
Separation and Identification of Desulpho Glucosinolates
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For quantitative analysis, separation of desulpho (DS)-GSL was conducted on a C18 column (150 × 3.0 mm i.d., 3 μm, Inertsil ODS-3; GL Sciences, Tokyo, Japan) using a HPLC system equipped with a diode array detector (Agilent Technologies, CA, USA). The UV–Visible detector wavelength was set at 227 nm. The elution solvent consisted of solvent A (ultra-pure water) and solvent B (acetonitrile). The samples were run for 40 min to separate entire compounds. The gradient solvent system used for the HPLC separation is mentioned in our previous report (Chun et al., 2013 ). For MS analysis, the eluate was diverted to an API 4000 Q TRAP tandem mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with electrospray ionization source in positive ion mode. The spray voltage was set to 5.5 kV and the capillary temperature was set to 550 °C. The scan of the masses ranged from m/z 100 to m/z 800.
6
Leaf Extract Metabolite Analysis
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The leaf extract was analyzed using the LCMS-8040 (Shimadzu). Mass spectra were acquired over a range of m/z 50–1000 using the Q3 scan mode. The solution was injected onto an Inertsil ODS-3 (250 × 2.1 mm, 5 µm, GL Sciences, Tokyo Japan) at a column temperature at 40 °C using a gradient of (A) 0.1% formic acid and (B) acetonitrile/water (80/20) containing 0.1% formic acid. The following gradient with a flow rate of 0.2 mL/min was used: 0–100% B (0–45 min), 100% B (45–50 min), and 0% B (50–60 min). Compounds were putatively identified by matching the experimental m/z values to the library of theoretical calculated m/z values in databases, including the Human Metabolome Database and the METLIN database.
7
Quantitative Glycopeptide Analysis by LC-MS/MS
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SRM analysis by LC-MS/MS was conducted with Dionex HPLC and AB Sciex 4000Q Trap® TurboIonSpray systems. Separation was performed with an LPG-3x00 pump, WPS-3000 auto sampler, FLM-3100 column component, and WVD-3400 detector under the control of the software Chromeleon 6.80 on Inertsil ODS-3 (2.1 × 150 mm, GL Sciences Inc). All data were analyzed by a series of software, Analyst 1.5 and MultiQuant 1.1.0.26. In MS/MS and LC-MS/MS analyses, measurements were performed by appropriate modes under the recommended default conditions as follows: (a) determination of the Q1 channel, enhanced mass mode and enhanced resolution mode; (b) determination of Q2 parameters, compound optimization mode; (c) determination of Q3 channels, enhanced product ion mode; (d) SRM/MRM assay, scheduled MRM mode; (e) LC conditions, multi-step gradient [(A) 0.1% FA aq. (B) 0.1% FA in CAN, 0 min: (A)/(B) = 90/10 → 45 min: 75/25 → 46 min: 10/90 → 50 min: 10/90 → 50.1 min: 90/10 → 60 min: 90/10]; column temperature, 25 °C; and injection volume, 10.0 µL. The SRM experiments were performed at a scan time of 0.10 s, dwell time of 20 ms, and peak width of 0.20 FWHM for glycopeptide 1 and 0.21 FWHM for glycopeptide 2.
8
Photochemical Degradation of Pc 3 Compound
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A 1 μM solution
ofPc 3 in PBS buffer (pH 7.5) with 1 mM NaAA was irradiated
with excitation light of a spectrofluorometer FP-8600 (676.5 nm, 3.5
mW cm–2) for 30 or 60 min. HPLC analyses of the
photolysis were performed by an HPLC system (Shimadzu Corporation)
with a reverse-phase column Inertsil ODS-3 (4.6 mm × 250 mm)
(GL Sciences Inc.), using eluent A (H2O, 0.1 M TEAA) and
eluent B (99% MeCN, 1% H2O) (A/B = 80/20 to 0/100 in 10
min; flow rate, 1.0 mL min–1). The products by photolysis
were isolated and characterized with NMR and MS (ESI and MALDI-TOF).
Degradate A. 1H NMR (400 MHz, DMSO-d6): δ −2.93 (s, 6H), −2.48 (s, 1H),
−2.41 to −2.35 (m, 2H), −1.07 to −0.95
(m, 2H), 1.37–1.48 (m, 6H), 1.83–1.91 (m, 2H), 2.25–2.34
(m, 12H), 8.47 (dd, 8H, J = 5.6, 3.1 Hz), 9.69 (dd,
8H, J = 5.6, 3.1 Hz). MS (ESI+) m/z: [M+TEA]+ as TEA salt, calcd
for C52H63N10Na2O11S3Si2, 1201.3; found, 1201.3.
Degradate B. MS (MALDI+) m/z: [M + H]+ calcd for C32H19N8O2Si, 575.1; found, 574.7.
of
with excitation light of a spectrofluorometer FP-8600 (676.5 nm, 3.5
mW cm–2) for 30 or 60 min. HPLC analyses of the
photolysis were performed by an HPLC system (Shimadzu Corporation)
with a reverse-phase column Inertsil ODS-3 (4.6 mm × 250 mm)
(GL Sciences Inc.), using eluent A (H2O, 0.1 M TEAA) and
eluent B (99% MeCN, 1% H2O) (A/B = 80/20 to 0/100 in 10
min; flow rate, 1.0 mL min–1). The products by photolysis
were isolated and characterized with NMR and MS (ESI and MALDI-TOF).
Degradate A. 1H NMR (400 MHz, DMSO-d6): δ −2.93 (s, 6H), −2.48 (s, 1H),
−2.41 to −2.35 (m, 2H), −1.07 to −0.95
(m, 2H), 1.37–1.48 (m, 6H), 1.83–1.91 (m, 2H), 2.25–2.34
(m, 12H), 8.47 (dd, 8H, J = 5.6, 3.1 Hz), 9.69 (dd,
8H, J = 5.6, 3.1 Hz). MS (ESI+) m/z: [M+TEA]+ as TEA salt, calcd
for C52H63N10Na2O11S3Si2, 1201.3; found, 1201.3.
Degradate B. MS (MALDI+) m/z: [M + H]+ calcd for C32H19N8O2Si, 575.1; found, 574.7.
9
HPLC Determination of Caffeine Concentration
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The concentration of caffeine was determined using a high-performance liquid chromatography (HPLC) system (Prominence, Shimadzu Corporation, Kyoto, Japan) equipped with a UV detector (SPD-M20A, Shimadzu Corporation). Briefly, the receiving solutions were mixed with the same volume of methanol. After centrifugation at 21,500 ×g at 4°C for 5 min, the resulting supernatant (20 μL) was injected directly into the HPLC system. Chromatographic separation was performed at 40°C using an Inertsil ODS-3, 5 μm in diameter, 4.6 mm I.D. × 150 mm (GL Sciences Inc., Tokyo, Japan). The mobile phase was 0.1% phosphoric acid : methanol 7 : 3 v/v and the flow rate was 1.0 mL/min. UV absorbance detection was performed at 280 nm.
10
HPLC Determination of Cardiovascular Drugs
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Concentrations of drugs (LC, ISDN, AMP, IP, BP and ISMN) in the samples were determined using an HPLC system (Prominence; Shimadzu, Kyoto, Japan) equipped with a UV detector (SPD-M20A; Shimadzu, Kyoto, Japan). The drug samples (0.2 mL) were added to the same volume of acetonitrile for ISMN or acetonitrile containing internal standard (methylparaben for LC, butylparaben for ISDN, AMP and IP, and propylparaben for BP), and mixed with a vortex mixer. After centrifugation at 21,500× g and 4 °C for 5 min, 20 μL of the supernatant was injected into the HPLC system. Chromatographic separation was performed using an Inertsil-ODS-3 (5 μm, 150 × 4.6 mm2 i.d.; GL Science, Kyoto, Japan) at 40 °C. The mobile phase was 0.1% phosphoric acid containing 5 mM sodium 1-heptanesulfate/acetonitrile (70/30, v/v) for LC, water/acetonitrile (55/45, v/v) for ISDN, 0.1% phosphoric acid containing 5 mM sodium dodecyl sulfate/acetonitrile (30/70, v/v) for AMP, 0.1% phosphoric acid/acetonitrile (55/45, v/v) for ISMN and water/acetonitrile (90/10, v/v) for ISMN. The flow rate was adjusted to 1.0 mL/min and detection was performed at UV 220 nm (ISMN and ISDN), 230 nm (LC), 245 nm (AMP), 260 nm (BP) or 263 nm (IP).
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