The relative molecular mass (Mr) distribution of gluten proteins was determined using gel permeation (GP)-HPLC. GP-HPLC analysis was performed using a Shimadzu Nexera XS HPLC (Shimadzu, Nakagyo-ku, Kyoto, Japan). The column was a BioSep-SEC-s3000 (300 × 4.6 mm, 5 µm, Phenomenex, Aschaffenburg, Germany) operated at 22 °C. The mobile phase consisted of A: 0.1% TFA in bidist. water and B: 0.1% TFA in acetonitrile. The flow rate was 0.3 ml/min with an isocratic elution (50% A, 50% B). Detection was performed using a DAD at 210 nm. Proteins with known Mr were used to determine the integration limits for specific Mr ranges. The proteins used were cytochrome C from horse heart (12.4 kDa), carbonic anhydrase from bovine erythrocytes (29 kDa) and albumin from bovine serum (66 kDa). The Mr ranges were the following: (1) > 66 kDa; (2) 66–29 kDa; (3) 29–12.4 kDa; and (4) < 12.4 kDa. In each range, the area under the curve (AUC) was integrated and calculated as a percentage of the total area.
Biosep sec s 3000
Manufactured by Phenomenex
Sourced in United States
The BioSep-SEC-S 3000 is a size exclusion chromatography column designed for the separation and analysis of biological macromolecules. It features a silica-based stationary phase and is suitable for use with aqueous mobile phases.
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Lab products found in correlation
Ag1 x8, by Bio-Rad (1 mentions)
Mouse serum, by Thermo Fisher Scientific (1 mentions)
Polygram sil g uv254 plates, by Macherey-Nagel (1 mentions)
Astra 5 software, by Wyatt Technology (1 mentions)
Hplc system, by Wyatt Technology (1 mentions)
Dawn heleos 2, by Wyatt Technology (1 mentions)
Optilab rex, by Wyatt Technology (1 mentions)
Empower 2, by Waters Corporation (1 mentions)
2535 quaternary gradient module pump, by Waters Corporation (1 mentions)
Amicon ultra 15 filter, by Merck Group (1 mentions)
Zorbax eclipse plus, by Agilent Technologies (1 mentions)
Dionex ics 3000 hplc system, by Thermo Fisher Scientific (1 mentions)
Biosep sec s2000, by Phenomenex (1 mentions)
Econofilter, by Agilent Technologies (1 mentions)
Securityguardtm, by Phenomenex (1 mentions)
Kj0 4282, by Phenomenex (1 mentions)
Lc10 hplc, by Shimadzu (1 mentions)
Biosep sec s4000, by Phenomenex (1 mentions)
12 protocols using biosep sec s 3000
1
Gel Permeation HPLC Analysis of Gluten Proteins
2
Radiolabeling and Quality Control of 64Cu-Conjugated Antibodies
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64Cu was produced in a PETtrace cyclotron (16 MeV; GE Medical Systems, Uppsala, Sweden) by proton irradiation of 64Ni on a platinum/iridium plate (90/10) (30 mg, > 95% enrichment; Chemotrade Chemiehandelsgesellschaft mbH, Duesseldorf, Germany) and separated from metallic impurities by ion-exchange chromatography (AG1x8, Bio-Rad Laboratories, Hercules, CA, USA). Radiolabeling was performed according to a modification of a procedure that has been described in the literature 29 (link). Briefly, chelator-conjugated antibodies were incubated with 0.2-1 MBq [64Cu]CuCl2 per µg for 1 h at 42 °C in 0.25 M sodium acetate buffer. Quality control was performed by thin-layer chromatography (TLC) using Polygram SIL G/UV254 plates (Macherey-Nagel, Dueren, Germany) and citrate buffer (tracer remained at baseline, observed Rf for unbound 64Cu of 0.2-0.8). High-performance size-exclusion chromatography (HPSEC, BioSep SEC-s3000, Phenomenex; saline sodium citrate buffer, 1 ml/min) was used to check for fragmentation and aggregation of the antibodies.
To assess the release of 64Cu from the radiotracer, the radioimmunoconjugates were incubated with PBS, mouse serum (Life Technologies, Carlsbad, CA, USA), or in presence of a competitor (ethylenediaminetetraacetic acid (EDTA); Sigma-Aldrich) at 37 °C for a period of 2 days. TLC was performed repetitively (n = 1-3).
To assess the release of 64Cu from the radiotracer, the radioimmunoconjugates were incubated with PBS, mouse serum (Life Technologies, Carlsbad, CA, USA), or in presence of a competitor (ethylenediaminetetraacetic acid (EDTA); Sigma-Aldrich) at 37 °C for a period of 2 days. TLC was performed repetitively (n = 1-3).
3
SEC-MALLS Characterization of Protein Complexes
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SEC-MALLS experiments were performed on a Shimadzu HPLC system with the SPD20A UV/Vis detector linked to a Wyatt Dawn HELEOS-II 18-angle light-scattering detector and a Wyatt Optilab rEX refractive index monitor. SEC was carried out on a Phenomenex HPLC column BioSep-SEC-S 3000, equilibrated and run at a flow rate of 1 ml/min in a solution consisting of 25 mM HEPES, pH 7.5, 200 mM NaCl and 2 mM DTT. 100-μl samples of individual proteins (APE1, Polβ, and XRCC1) at concentrations ranging from 2.1 to 4.2 mg/ml or equimolar mixtures of two (or three) proteins were incubated at 4°C for 2 h and injected using an SIL-20A Autosampler. The SEC-MALLS experiments were performed at room temperature. The profile line was monitored using a UV monitor at 280 nm and a refractive index detector at 690 nm. The data were analyzed with the Astra V software (Wyatt Technology) using a refractive index increment (dn/dc) value of 0.190.
4
Hb Binding and Pharmacokinetics Following Hp Infusion
5
Protein Molecular Integrity Analysis
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Molecular integrity was evaluated by using a BioSep-SEC-s3000 (600 × 7.5 mm) column (Phenomenex, Torrance, CA, USA) connected to a Waters 2535 Quaternary Gradient Module pump and Waters 2998 Photodioide Array Detector controlled by a Waters 600 controller using Empower 2 software (Waters, Milford, MA, USA). Mobile phase: 1 mM potassium phosphate (K2HPO4/KH2PO4) pH 7.4. The injection volume of each protein solution was 50 μL. Detection was performed at three wavelengths: 280, 404, and 414 nm.
6
Antibody Conjugation with NODAGA Chelator
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Protein A-purified mAb MC3 was buffered with 0.1 M sodium carbonate (pH 9, Chelex-treated) using an Amicon Ultra-15 filter unit (30 kDa MWCO, Merck Millipore, Darmstadt, Germany). Twenty molar equivalents of chelator p-NCS-Bz-NODAGA (Chematech, Dijon, France) were reacted with the antibody for 60 min at room temperature, with subsequent antibody purification via an Amicon Ultra-15 filter using Chelex-treated 0.25 M sodium acetate (pH 6). This protocol typically yields 1–3 chelators per antibody. Elution profile in High Pressure Size Exclusion Chromatography (HPSEC, Phenomenex BioSep SEC-s3000 300 × 4.6 mm, 1.5 mL/min PBS with 50 mM EDTA) was not discernible from the unconjugated antibody.
7
HPLC Analysis of TA and OVA Drugs
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Analysis of TA samples was carried out using reversed-phase Agilent 1260 Infinity Quaternary System HPLC using an Agilent Zorbax Eclipse Plus 250 mm, 4.6 mm ID, 5 μm particle size, C18 bonded silica column and an Agilent Zorbax guard column held at 25°C (Agilent Technologies Ltd., Stockport, UK). A mobile phase of 60% water and 40% acetonitrile at UV absorbance of 236 nm was found to give optimal peak shape. Injection volume was fixed to 20 μl and flow rate was set at 0.8 mL/min. A standard calibration curve was then prepared, between the concentration range of 0.1–250 μg/mL, and used to determine the TA drug concentration in each of the release samples.
Analysis of OVA samples was carried out using Agilent 1260 Infinity Quaternary System HPLC using a Phenomenex BIOSEP-SEC-s3000 300 mm, 7.8 mm ID, 3 μm particle size column and GFC 3000 guard column held at 25°C (Phenomenex., Cheshire, UK). A mobile phase of 250 mM NaCl at UV absorbance of 214 nm was found to give optimal peak shape. Injection volume was fixed to 20 μL and flow rate was set at 1 mL/min. A standard calibration curve was then prepared, between the concentration range 3.125–100 μg/ml, and used to determine the OVA drug concentration in each of the release samples.
Analysis of OVA samples was carried out using Agilent 1260 Infinity Quaternary System HPLC using a Phenomenex BIOSEP-SEC-s3000 300 mm, 7.8 mm ID, 3 μm particle size column and GFC 3000 guard column held at 25°C (Phenomenex., Cheshire, UK). A mobile phase of 250 mM NaCl at UV absorbance of 214 nm was found to give optimal peak shape. Injection volume was fixed to 20 μL and flow rate was set at 1 mL/min. A standard calibration curve was then prepared, between the concentration range 3.125–100 μg/ml, and used to determine the OVA drug concentration in each of the release samples.
8
Size Exclusion Chromatography of Protein Samples
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BSA and BSA-Tpp samples containing 40 μg of protein were suspended in 25 mM phosphate buffer (pH 6.8) and centrifuged at 10 000 × g. The supernatant was transferred into HPLC vials and placed into cooled (4 °C) auto sampler chamber of HPLC system.
Size exclusion chromatography was performed using Dionex ICS-3000HPLC system (Thermo Scientific, USA) and the BioSep-SEC-S3000 (Phenomenex, 5 μm × 400 Å, 600 × 7.8 mm) column. The proteins were eluted at a flow rate 0.5 ml min−1 at 20 °C in isocratic 25 mM phosphate buffer (pH 6.8) containing 100 mM NaCl during 60 min. The column eluate was monitored by PDA detector at 280 and 420 nm with simultaneous spectra recording between 200 and 600 nm.
Size exclusion chromatography was performed using Dionex ICS-3000HPLC system (Thermo Scientific, USA) and the BioSep-SEC-S3000 (Phenomenex, 5 μm × 400 Å, 600 × 7.8 mm) column. The proteins were eluted at a flow rate 0.5 ml min−1 at 20 °C in isocratic 25 mM phosphate buffer (pH 6.8) containing 100 mM NaCl during 60 min. The column eluate was monitored by PDA detector at 280 and 420 nm with simultaneous spectra recording between 200 and 600 nm.
9
Radiolabeled Transferrin Serum Stability
10
SOD1 Oligomerization Assay in Lipid Modulation
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SOD1 WT (10 µM) in apo and holo forms were incubated in 50 mM phosphate buffer pH 7.4 containing 150 mM NaCl and 100 µM DTPA for 24 h at 37 °C in the presence of 250 µM cholesterol, cholesterol hydroperoxides, Seco A or Seco B. Docosahexaenoic acid (DHA, 250 uM) was used as a positive control [33] . For detection of SOD1 oligomers, SDS-PAGE was performed under reducing (+β-mercaptoethanol) and non-reducing (-β-mercaptoethanol) conditions in a 12% polyacrylamide gel. Aliquots of the samples (20 µL) were incubated in sample buffer (62 mM Tris-HCl, pH 6.8 containing 10% glycerol, 2% SDS, 0.01% bromophenol blue) in the absence and presence of β-mercaptoethanol (200 mM) for 5 min at 95 °C and then applied on the gel. Silver nitrate was applied for gel staining. Size exclusion chromatography (SEC) was performed using the column BioSep-SEC-S3000 (300 ×7.8 mm, Phenomenex, USA). Each sample was eluted with 50 mM phosphate buffer, pH 7.4 containing 150 mM NaCl. Fluorescence detector conditions were: excitation wavelength at 280 nm and emission at 340 nm.
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