The experiments were performed on a HP3D CE system (Agilent Technologies Waldbronn, Germany). This instrument is equipped with an autosampler and a temperature control system (15–60 °C ± 0.1 °C). The detection was carried out using an on-column diode array detector. UV detection was set at 280 nm. Bare fused-silica capillaries with an internal diameter of 50 μm were purchased from Optronis (Kehl, Germany). Capillaries of 48.5 cm total length (8.5 cm effective length) were dynamically coated with poly (ethylene oxide) (PEO) according to the procedure previously reported by [57 (link)]. Experiments were performed using the outlet injection mode. VLP samples were injected hydrodynamically by applying a pressure of − 50 mbar during 15 s. Anti-GFLV antibody (DSMZ) and mouse monoclonal antibody raised against amino acids 1–40 of HPV16 L2 (Santa Cruz) were used for affinity experiments. The separation was performed applying a voltage of 10 kV (normal polarity mode) and the capillary was thermostated at a temperature of 15 °C.
Hp 3d ce system
Manufactured by Agilent Technologies
Sourced in Germany
The HP 3D CE system is a laboratory equipment designed for electrophoretic separation and analysis of biomolecules. It provides capabilities for high-resolution separation and detection of DNA, proteins, and other macromolecules.
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Lab products found in correlation
Ts 100 thermo shaker, by Biosan (1 mentions)
Msd ion trap mass spectrometer, by Agilent Technologies (1 mentions)
2002 potentiometer, by Crison (1 mentions)
Electrode 52 03, by Crison (1 mentions)
6220 oa tof lc ms mass spectrometer, by Agilent Technologies (1 mentions)
Chemstation software, by Agilent Technologies (1 mentions)
Masshunter workstation software, by Agilent Technologies (1 mentions)
5 protocols using hp 3d ce system
1
Capillary Electrophoresis of Virus-Like Particles
2
CE-MS Analysis of Biomolecule Samples
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All experiments were performed on an Agilent Technologies HP 3D CE system (Waldbronn, Germany) coupled to an MSD Ion Trap mass spectrometer (Agilent Technologies). An electrospray G1603A Agilent Technologies sprayer was used as a sheath-flow-CE-ESI-MS interface. The sheath liquid was delivered by an infusion pump KD Scientific 100 Series (Holliston, MA, USA). The detailed conditions affecting CE-MS have been discussed elsewhere, as has the tuning of the IT mass spectrometer [33] (link). pH measurements were performed with a Crison 2002 potentiometer and a Crison electrode 52-03 (Crison Instruments, Barcelona, Spain). Sample incubation was performed with a Thermo-Shaker TS-100 (Biosan, Warren, USA). Centrifugation was performed in a thermostated Rotanta 460 centrifuge (Hettich Zentrifugen, Tuttlingen, Germany).
3
CE-MS Optimization for Glycopeptide Analysis
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CE-MS experiments were performed in a HP 3D CE system coupled to a 6220 oa-TOF LC/MS mass spectrometer with an orthogonal sheathflow interface (Agilent Technologies). The sheath liquid (50:50 (v/v) iPrOH/H2O with 0.05% (v/v) of HFor) was delivered at a flow rate of 3.3 µL•min -1 by a KD Scientific 100 series infusion pump (Holliston, MS ,USA) and degassed for 10 min by sonication before use. CE control and separation data acquisition (e.g. voltage, temperature and current) were performed using Chemstation software (Agilent Technologies) that was running in combination with the MassHunter workstation software (Agilent Technologies) for control, data acquisition and processing of the mass spectrometer. The mass spectrometer was tuned and calibrated following the manufacturer's instructions. A "check tune" of the instrument was performed every day in positive mode to ensure accurate mass assignments. Instrumental parameters were optimized for the analysis of rhEPO O126-and N83-glycopeptides in a previous study [18] (link). The optimized operational conditions in positive electrospray ionization (ESI) mode were: capillary voltage 4000 V, drying gas (N2) temperature 200 ºC, drying gas flow rate 4 L min -1 , nebulizer gas (N2) 10 psig, fragmentor voltage 190 V, skimmer voltage 60 V and OCT 1 RF Vpp voltage 300 V. Data were collected in profile (continuum) at 1 spectrum•s -1 (approx.
4
CE-based Cation and Anion Separation
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CE experiments were carried out with an HP 3D CE system (Agilent Technologies, Santa Clara, CA) equipped with an eDAQ C 4 D system (eDAQ, Denistone East, Australia). CE ChemStation (Agilent) was used for CE control, and data acquisition was done by eDAQ chart. In all CE separations, fused silica capillaries (GL Sciences, Tokyo, Japan) of 25 μm i.d. with an effective length of 20 cm for cations and 15 cm for anions, respectively (35 cm in total), were used with 30 mM malic acid containing 100 mM DDAPS, 3 mM of 18-crown-6ether and 18 mM L-histidine (pH 3.6) as BGE. The capillary was thermostated at 25°C. The buffer concentration in the sample solution was made up by mixing with concentrated BGE solution. The sample in BGE solution was introduced from the anodic end for 2 min at 3.45 kPa for filling the capillary. Separation was conducted with an applied voltage of 30 kV. After CE, the capillary was conditioned, in turn, with 0.1 M NaOH for 5 min, 50% (v/v) MeOH for 5 min, water for 3 min and BGE for 5 min. C 4 D detector settings were as follows: low pass = 2 Hz; frequency = 800 kHz; amplitude = 70%; headstage gain = ON.
5
Capillary Electrophoresis of Biomolecules
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The CE experiments were performed on a HP 3D CE system (Agilent Technologies, Waldbronn, Germany) equipped with an on-column UV-vis DAD and an air thermostating system. The CE instrument was driven and the data were collected by the software 3D CE ChemStation Rev.09.01 (Agilent Technologies). Uncoated fused silica capillaries (50 m inner diameter, 365 m outer diameter, total length 48.5 cm, effective length 40.0 cm) were used for running the analyses and were purchased from Unifibre (Settimo Milanese, Italy). The detection wavelength was 220 nm and the samples were hydrodynamically injected (50 mbar for 3 s), followed by a BGE plug at 50 mbar for 3 s. Each new capillary was flushed with 1 M NaOH, 0.1 M NaOH and water (5 min each). Between injections, the capillary was washed with methanol, 0.1 M NaOH, water for 1 min each, and then with the proper BGE for 3 min. The working conditions (with the interval corresponding to the MODR) were: temperature, 18
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