NMR experiments were carried out on a 500 MHz Bruker Avance III HD using a 5 mm BBFO RT probe equipped with a Z gradient. A series of 1D 1H and 2D 1H–1H homonuclear (COSY, TOCSY, and NOESY) or 1H–13C heteronuclear (HSQC and HSQC-NOESY) NMR spectra were recorded. All the experiments were carried out at 50 °C in 5 mm NMR tubes (VWR International) using the solvent deuterium oxide (D2O) (D 99.90%, Cambridge Isotope Laboratories, Inc.). The acquired NMR data were further processed and analyzed using MestreNova 14.1.0 and TopSpin 4.0 software. For HPSEC-MALS, an Ultimate 3000 high-performance liquid chromatography system (Thermo Scientific, Sunnyvale, CA, USA) connected to the SEC column (Acquity BEH SEC column, 200 Å, 1.7 μm, 4.6 mm × 300 mm, Waters, MA, USA) was used. The eluent was monitored by MALS using a DAWN HELEOS II MALS detector (Wyatt Technologies Co., Santa Barbara, CA, USA). Ammonium acetate and high-performance liquid chromatography grade water were purchased from Fisher Scientific (Fair Lawn, NJ, USA). SPR measurements were performed on a BIAcore 3000, operated using 3000 control and Biaevaluation software, version 4.0.1. (Uppsala, Sweden). Sensor streptavidin (SA) chips were procured from Cytiva (Uppsala, Sweden).
Acquity beh sec column
Manufactured by Waters Corporation
Sourced in United States
The Acquity BEH SEC column is a size exclusion chromatography (SEC) column designed for the analysis of macromolecules, such as proteins and polymers. It utilizes Waters' Bridged Ethylsiloxane/Silica Hybrid (BEH) technology to provide high-performance separation and resolution. The column is suitable for a range of applications that require the characterization of molecular weight and size distribution of samples.
Automatically generated - may contain errors
Lab products found in correlation
Ammonium acetate, by Thermo Fisher Scientific (1 mentions)
Streptavidin sa sensor chips, by Cytiva (1 mentions)
Nmr tubes, by Avantor (1 mentions)
Avance 3 hd, by Bruker (1 mentions)
Agilent 1290 infinity 2 uhplc system, by Agilent Technologies (1 mentions)
Compass dataanalysis 4.4 sr1, by Bruker (1 mentions)
Maxis 2 q tof instrument, by Bruker (1 mentions)
Ultimate 3000 uhplc system, by Thermo Fisher Scientific (1 mentions)
3 protocols using acquity beh sec column
1
NMR and HPSEC-MALS Characterization of Biomolecules
2
Characterization of Fab-Albumin Complexes
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To characterize the nature of Fab–albumin complex formation in vivo, SEC studies were performed with the vitreous humor samples obtained in the rabbit study. As outlined in Table 2 , FabA and FabB were also incubated with HSA and CSA in PBS, as well as in human and cynomolgus monkey VH at 37 °C for 18 h. The samples were stored at −20 °C prior to SEC analysis.
Protein separation was carried out by SEC on an Agilent 1290 Infinity II UHPLC system (Agilent Technologies Inc, Santa Clara, CA, USA) using an Acquity BEH SEC column (200 Å, 1.7 × 100 mm, Waters Corporation, Milford, MA, USA) with 100 mM sodium phosphate buffer at pH 7.4 containing 10% ethanol as mobile phase. The method run time was 10 min at 250 µL/min isocratic flow. Fractions were collected between 4 and 10 min for 0.05 min (12.5 µL) in a Deepwell LumaPlate384 (Perkin Elmer, Waltham, MA, USA) and radioactivity was measured on a TopCount NXT HTS Microplate Counter (Perkin Elmer, Waltham, MA, USA).
Protein separation was carried out by SEC on an Agilent 1290 Infinity II UHPLC system (Agilent Technologies Inc, Santa Clara, CA, USA) using an Acquity BEH SEC column (200 Å, 1.7 × 100 mm, Waters Corporation, Milford, MA, USA) with 100 mM sodium phosphate buffer at pH 7.4 containing 10% ethanol as mobile phase. The method run time was 10 min at 250 µL/min isocratic flow. Fractions were collected between 4 and 10 min for 0.05 min (12.5 µL) in a Deepwell LumaPlate384 (Perkin Elmer, Waltham, MA, USA) and radioactivity was measured on a TopCount NXT HTS Microplate Counter (Perkin Elmer, Waltham, MA, USA).
3
Characterization of FabA–HSA Complex
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FabA–HSA complex composition was characterized by high-resolution intact mass spectrometry. Protein separation was carried out by SEC on an Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) using an Acquity BEH SEC column (200 Å, 1.7 × 100 mm; Waters Corporation, Milford, MA, USA) with 100 mM ammonium acetate pH 6.8 as mobile phase. The method run time was 10 min at 250 µL/min isocratic flow. Intact mass analysis was performed on a maXis II qToF instrument (Bruker Corporation, Billerica, MA, USA). The ion polarity was set to positive mode, covering the mass range of 800–10,000 m/z. Spectra were recorded at 0.5 Hz. Capillary voltage was 4500 V at a nebulizer pressure of 2 bar and a dry gas flow rate of 10 L/min at 280 °C. In-source fragmentation was enabled at 30 eV. The raw files were analyzed using Compass DataAnalysis 4.4 SR1 (Bruker Corporation, Billerica, MA, USA) and PMI-Intact v3.8-11 x64 (Protein Metrics Inc., San Carlos, CA, USA).
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