Purified hGHR-GFP in detergent was desalted and concentrated using C4 ZipTips (Merck Millipore), eluted into 50% acetonitrile/0.1% formic acid, and directly infused into an Orbitrap Fusion Tribrid mass spectrometer equipped with an offline nanospray source using borosilicate capillaries (Thermo Scientific). The capillary voltage was 1.5 kV in positive ionization mode, and the pressure in the ion-routing multipole was maintained at 0.11 torr. Ten percent of HCD activation energy in the ion trap was used to dissociate any residual detergents from the protein. Spectra were acquired in the Orbitrap mass analyzer operated in high mass mode between 1500 to 6000 mass/charge ratio (m/z) with an injection time of 10 ms and a resolution of 60,000 full width at half maximum at 200 m/z. Data were analyzed using Excalibur (Thermo Scientific) and UniDec (unidec.chem.ox.ac.uk ) software packages.
Excalibur
Manufactured by Thermo Fisher Scientific
Sourced in United States, Italy
The Excalibur is a high-performance laboratory centrifuge designed for a variety of applications. It features a robust and reliable construction, ensuring consistent and accurate results. The Excalibur is capable of handling a wide range of sample volumes and tube sizes, making it a versatile choice for various laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
Borosilicate capillaries, by Thermo Fisher Scientific (2 mentions)
Orbitrap fusion tribrid, by Thermo Fisher Scientific (2 mentions)
C4 ziptip, by Merck Group (1 mentions)
Prism 7, by GraphPad (1 mentions)
Gfp trap ma magnetic beads, by Proteintech (1 mentions)
Zeba spin column, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Hypersil gold c18 column, by Thermo Fisher Scientific (1 mentions)
Lcq fleet ion trap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Accela lc system, by Thermo Fisher Scientific (1 mentions)
Trace gc, by Thermo Fisher Scientific (1 mentions)
7 protocols using excalibur
1
Purified hGHR-GFP Characterization by MS
2
Peak Integration and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Thermo Excalibur software (2.2 SP1.48) was used for peak integration and quantification of data. GraphPad Prism 7 (GraphPad Software) was used for statistical analysis and illustrations.
3
GFP Purification from Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A modified transfection protocol was used for larger-scale GFP expression for bead purification, increasing the amount of total DNA to 6 μg (Mma PylT/RS AF and PylT/sfGFP150TAG at 2 + 8 ratio) per ml culture and transfecting 5.0–8.0 × 105 cells/ml with 2 μg of polyethylenimine (PEI) per μg of DNA. PazK was supplemented to 0.5 mM at transfection and until harvest after 6 days. Cells were lysed in RIPA buffer supplemented with 1× cOmplete protease inhibitor (Roche). The insoluble fraction was removed by centrifugation. Expressed GFP was captured on GFP-Trap_MA magnetic beads (ChromoTEK), washed with RIPA buffer and PBS, and eluted in 1% (v/v) acetic acid.
Purified GFP samples were desalted and rebuffered into 100 mM ammonium acetate, pH 7.5, using ZebaSpin columns with a 7-kDa cutoff (Thermo). Samples were directly infused into an Orbitrap Fusion Tribrid mass spectrometer equipped with an offline nanospray source using borosilicate capillaries (Thermo). The capillary voltage was 1.5 kV, and the pressure in the ion-routing multipole was maintained at 0.11 torr. Spectra were acquired in the Orbitrap mass analyzer operated in high mass mode at a resolution of 60.000 between 1,000 and 4,000 m/z. Data were analyzed using Excalibur (Thermo).
Purified GFP samples were desalted and rebuffered into 100 mM ammonium acetate, pH 7.5, using ZebaSpin columns with a 7-kDa cutoff (Thermo). Samples were directly infused into an Orbitrap Fusion Tribrid mass spectrometer equipped with an offline nanospray source using borosilicate capillaries (Thermo). The capillary voltage was 1.5 kV, and the pressure in the ion-routing multipole was maintained at 0.11 torr. Spectra were acquired in the Orbitrap mass analyzer operated in high mass mode at a resolution of 60.000 between 1,000 and 4,000 m/z. Data were analyzed using Excalibur (Thermo).
4
Quantitative Analysis of Decursin by HPLC-MS/MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantification of decursin was performed using an Accela LC system (Thermo Fisher Scientific; MA, USA) equipped with an autosampler, degasser, and quaternary solvent pump. Decursin and IS were separated on a Hypersil GOLD C18 column (2.1 mm × 100 mm, 1.9 μm; Thermo Fisher Scientific, MA, USA) at 35℃. The flow rate was set at 300 μL/min with an injection volume of 5 μL. The mobile phase consisted of water (containing 0.1% formic acid, A) and acetonitrile (B) and the gradient elution applied was as follows: 30–60% (B) for 0–4 min, 60% (B) for 0.5 min, and then re-equilibrated to 30% (B) until the end of the analysis.
An LCQ Fleet ion-trap mass spectrometer (Thermo Fisher Scientific; MA, USA) was used to detect compounds in the eluent using the electrospray ionization source in the positive-ion mode. MS conditions were as follows: sheath gas (nitrogen), 50 arbitrary units; auxiliary gas (nitrogen), 20 arbitrary units; spray voltage, 5.0 kV; capillary temperature, 300℃; and capillary voltage, 30.0 V. Quantification of the compound was performed in the selective ion monitoring mode at 329 m/z [M+H]+ for decursin and 415 m/z [M+H]+ for the IS. Data was processed using Excalibur (v. 2.1.0; Thermo Fisher Scientific, CA, USA).
An LCQ Fleet ion-trap mass spectrometer (Thermo Fisher Scientific; MA, USA) was used to detect compounds in the eluent using the electrospray ionization source in the positive-ion mode. MS conditions were as follows: sheath gas (nitrogen), 50 arbitrary units; auxiliary gas (nitrogen), 20 arbitrary units; spray voltage, 5.0 kV; capillary temperature, 300℃; and capillary voltage, 30.0 V. Quantification of the compound was performed in the selective ion monitoring mode at 329 m/z [M+H]+ for decursin and 415 m/z [M+H]+ for the IS. Data was processed using Excalibur (v. 2.1.0; Thermo Fisher Scientific, CA, USA).
5
PAH Quantification via GC-MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Individual PAHs were characterized using a gas chromatograph equipped with a mass spectrometer (Trace-GC and Trace Q MS) and controlled by the proprietary software Excalibur (all from Thermo Fisher, Rodano MI, Italy). The analytes were separated applying a temperature gradient from 90 up to 290°C to a 25-m-long RT5MS type column (i.d. = 250 μm, film thickness = 0.33 μm, Superchrom, Milan, Italy), under a Helium constant flow of 1.0 mL.min-1. For identification, the combination of relative retention times, mass spectra and ion trace ratios of the peaks was compared with that of authentic PAH standards. For quantitative purposes, the peak area of each compound had compared with that of its perdeuterated homologue or the closest internal reference in the chromatogram (isotopic dilution method). The quantitative data were kept as reliable when the resulting concentrations lied within the operating ranges of the detector, i.e., 3.3 to ~ 1000 times the respective detection limits.
Filter blanks were included in the chromatograms in the correspondence; in the cases of phenanthrene and pyrene (light PAH congeners), blanks were quite important and accounted for in the quantitative determinations. The recovery rates varied between 83% and 106% (±9%), and the accuracy was better than 11% for all species.
Filter blanks were included in the chromatograms in the correspondence; in the cases of phenanthrene and pyrene (light PAH congeners), blanks were quite important and accounted for in the quantitative determinations. The recovery rates varied between 83% and 106% (±9%), and the accuracy was better than 11% for all species.
6
HPLC-UV/HRMS Analysis of Sophoricoside
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The analysis of sophoricoside was performed by a modified HPLC method described in the monograph of Sophorae Fructus in the Hong Kong Chinese Materia Medica Standards (Chinese Medicine Division, Department of Health 2012 ).
HPLC-UV/HRMS was performed with the same instrument configuration as described above. The mobile phase consisted of 0.4% aqueous (ultra-power water) formic acid (phase A) and acetonitrile: methanol (50:50 v/v) LC-MS grade with 0.4% formic acid (phase B). At a flow rate of 0.2 mL/min, the linear gradient was as follows: 0.00–40.00 min, 85–15% (A–B%) to 70–30% (A–B%) followed by a 5 min column wash with 15–85% (A–B%) and 5 min equilibration period with 85–15% (A–B%). UV detection wavelength of 254 nm, a column temperature of 40 °C, and an injection volume of 2 µL were applied.
MS parameters in the positive ionization mode were: ionization voltage 3500 V, ESI, ion transfer tube temperature 350 °C, vaporizer temperature 350 °C, 3 scans, resolution of 30000, HCD collision energy (%) 50. System control and data evaluation were performed with Thermo® Excalibur for LC-MS.
HPLC-UV/HRMS was performed with the same instrument configuration as described above. The mobile phase consisted of 0.4% aqueous (ultra-power water) formic acid (phase A) and acetonitrile: methanol (50:50 v/v) LC-MS grade with 0.4% formic acid (phase B). At a flow rate of 0.2 mL/min, the linear gradient was as follows: 0.00–40.00 min, 85–15% (A–B%) to 70–30% (A–B%) followed by a 5 min column wash with 15–85% (A–B%) and 5 min equilibration period with 85–15% (A–B%). UV detection wavelength of 254 nm, a column temperature of 40 °C, and an injection volume of 2 µL were applied.
MS parameters in the positive ionization mode were: ionization voltage 3500 V, ESI, ion transfer tube temperature 350 °C, vaporizer temperature 350 °C, 3 scans, resolution of 30000, HCD collision energy (%) 50. System control and data evaluation were performed with Thermo® Excalibur for LC-MS.
7
Mass Spectrometry Data Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!