Preparative separation of the mAb charge variants was carried out on a 9 × 250 mm ProPac™ WCX‐10 (Thermo Fisher Scientific Inc.) using an ÄKTA avant 25 and UNICORN 7.0 (GE Healthcare GmbH). mAb1 fractions were eluted using a linear NaCl gradient and pooled to obtain the charge variants as shown in Figure S1. As a control, all fractions of one run were pooled to obtain a sample of all charge variants exposed to the same conditions as the fractions. The charge variants and control sample were concentrated to around 2–5 g/L and rebuffered to 10 mM sodium phosphate pH 6.8 using 10 kDa molecular weight cut‐off Amicon® Ultra‐15 centrifugal filter units (Sigma‐Aldrich Chemie GmbH). In total, the fractionation experiment was carried out twice, and all resulting samples were checked by weak cation exchange chromatography (WCX) and capillary electrophoresis (CE). Biophysical properties (circular dichroism [CD], nano differential scanning fluorimetry [nano‐DSF], size exclusion chromatography [SEC]) were determined from one run in duplicates.
Unicorn 7
Manufactured by GE Healthcare
UNICORN 7.0 is a software platform developed by GE Healthcare for the operation and control of chromatography instrumentation. It provides a user-friendly interface for the setup, execution, and analysis of chromatographic separations. The software supports a range of GE Healthcare's chromatography systems and enables the collection, processing, and management of experimental data.
Automatically generated - may contain errors
Lab products found in correlation
Propac wcx 10, by Thermo Fisher Scientific (1 mentions)
Kta avant 25, by GE Healthcare (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (1 mentions)
Superose 12 10 300 gl column, by GE Healthcare (1 mentions)
Kta pure, by GE Healthcare (1 mentions)
Gel filtration standard, by Bio-Rad (1 mentions)
Superdex 200 increase, by GE Healthcare (1 mentions)
Kta pure 25 l1, by GE Healthcare (1 mentions)
Superdex 75 increase 10 300 gl, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
9 protocols using unicorn 7
1
Fractionation and Characterization of mAb Charge Variants
2
Purification of Monoclonal Antibody mAb1
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mAb1 was purified from cell culture free fluid using an in‐house‐packed MabSelect SuRe column, operated on an ÄKTA avant 25 using UNICORN 7.0 (GE Healthcare GmbH). The protein was eluted in 50 mM acetate pH 3.5. The eluted product pool was neutralized to pH 5.6 and stored at –70 °C until further use.
3
Size-Exclusion Chromatography of LptDE
4
C. elegans Lysate Fractionation
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C. elegans lysate was prepared as described above for LC-MS/MS, and loaded onto a Superose 6 Increase 10/300 GL column. The column was equilibrated with Lysis buffer A and 1 ml fractions were collected using Unicorn 7.0 (GE Healthcare Life Sciences).
5
FPLC Purification of Protein Complexes
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A Superose 12 10/300 GL column (GE Healthcare) was connected to the AKTA FPLC system. WHRN NPDZ12, USH2A-CT(S), and ADGRV1-CT were mixed at the molar ratio of 1:1:1. 200–300 μl samples were filtered and loaded into the column. The chromatography profiles were analyzed using Unicorn 7.0 (GE Healthcare). The fractionation samples collected were applied to SDS-PAGE and Coomassie blue staining.
6
Analyzing GFP-CT Protein Status
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To determine their status in solution, 5 mg purified GFP-CT proteins were loaded onto Superdex 200 Increase (GE Healthcare) connected to ÄKTA Pure (GE Healthcare). Samples were eluted in a buffer [50 mM Tris-HCl (pH 8.0)] at the flow rate of 0.5 ml/min for 60 min. The data were collected and analyzed by the Unicorn 7 software (GE Healthcare). An equal amount of Gel Filtration Standard (Bio-rad) was analyzed under the same conditions as a marker.
7
Determining Dimeric State of Purified Proteins
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To determine their dimeric state in solution, an equal amount of purified apo-CPB, CPB CP, or CPB CP_v1 (see the purification method below) was loaded onto Superdex 75 Increase 10/300 GL (GE Healthcare) connected to ÄKTA Pure 25 L1 (GE Healthcare). Samples were eluted in a buffer [20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5% (v/v) glycerol, and 0.5 mM TCEP] at the flow rate of 0.5 mL/min for 60 min. The data were collected and analyzed by Unicorn 7 software (GE Healthcare). An equal amount of BSA (Sigma-Aldrich) was analyzed under the same conditions for comparison.
8
Determining PtmA2 Molecular Weight
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The molecular weight (MW) and monomeric state of PtmA2 in solution was determined by size-exclusion chromatography using a HiLoad 16/600 (16 × 600 mm) column (GE Healthcare) connected to a GE Healthcare ÄKTA pure HPLC system. The column was pre-equilibrated with two column volumes of 50 mM Tris, pH 8.0, containing 300 mM NaCl, and calibrated with ribonuclease A (13.7 kDa), ovalbumin (44 kDa), conalbumin (75 kDa), aldolase (158 kDa), and ferritin (440 kDa). The chromatography was carried out at 4 °C at a flow rate of 1 mL·min−1. Data analysis was performed using the Unicorn 7.0.2 software (GE Healthcare).
9
Determining PtmA2 Molecular Weight
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