Anti-TNC D IgG production is similar to previously described [10 (link), 18 (link)]. The monoclonal antibody targeting TNC D was purified via ProteinA through mediated capture of the Fc domains of the IgG. The clarified supernatant was purified on a AKTAPure FPLC (Cytiva, Marlborough, MA) via ProteinA affinity capture using a HiTrap MabSelect SuRe column (Cytiva, Marlborough, MA) and eluted at low pH. A second purification was performed using a Superdex 200 pg (Cytiva, Marlborough, MA) size exclusion column using tris-buffered saline to yield > 95% protein purity as determined by analytical liquid chromatography (Agilent, Santa Clara, CA). Endotoxin level was quantified to be < 1 EU/mg (Charles River Laboratories, Wilmington, MA).
Akta pure fplc
Manufactured by Cytiva
The AKTA Pure FPLC (Fast Protein Liquid Chromatography) is a laboratory instrument designed for protein purification and separation. It is capable of performing various chromatography techniques, including ion exchange, size exclusion, and affinity chromatography. The AKTA Pure FPLC is equipped with a user-friendly interface and advanced software for method development and data analysis.
Automatically generated - may contain errors
Lab products found in correlation
Mabselect sure, by Cytiva (3 mentions)
Akta pure, by Cytiva (3 mentions)
Superdex 200 pg, by Cytiva (2 mentions)
Endotoxin level, by Charles River Laboratories (2 mentions)
Hitrap mabselect sure column, by Cytiva (2 mentions)
Hitrap, by Cytiva (2 mentions)
Superdex 200 increase 10 300 gl column, by Cytiva (2 mentions)
Superdex 75 10 300 gl size exclusion column, by Cytiva (1 mentions)
Akta pure, by Wyatt Technology (1 mentions)
Minidawn treos mals detector, by Wyatt Technology (1 mentions)
Optilab t rex ri detector, by Wyatt Technology (1 mentions)
Dawn mals detector, by Wyatt Technology (1 mentions)
Dawn heleos 2, by Wyatt Technology (1 mentions)
Astra software, by Wyatt Technology (1 mentions)
Optilab t rex differential refractometer, by Wyatt Technology (1 mentions)
Mabselect sure prism column, by Cytiva (1 mentions)
Minidawn light scattering detector, by Wyatt Technology (1 mentions)
Optilab differential refractive index detector, by Wyatt Technology (1 mentions)
7 protocols using akta pure fplc
1
Purification of Anti-TNC D IgG
2
Oligomeric state analysis of SPACA6
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The oligomeric state of tag-removed, fully glycosylated SPACA6 was assessed by SEC-MALS. 0.14 mg Bovine Serum Albumin (BSA) and 0.14 mg SPACA6 were prepared in 1X PBS at a concentration of 1.2 mg mL−1. A Superdex 75 10/300 GL size-exclusion column (Cytiva/GE) was equilibrated overnight with 5 CV PBS. Monomeric BSA (MW = 66,432 Da) was used as a reference calibration standard. Prior to SEC-MALS analysis, each sample was centrifuged at 15,000 × g for 15 min at 4 °C and then the supernatant was loaded onto the size-exclusion column on an AKTA Pure FPLC (Cytiva) at 0.2 mL min−1. Triple detection was performed by measuring absorbance at 280 nm using the integrated UV monitor on the AKTA Pure, three-angle light scattering using the miniDAWN TREOS MALS detector (Wyatt) and refractive index (RI) using Optilab T-rEX RI detector (Wyatt). The data were processed, and weight-averaged molecular mass was calculated using the ASTRA software package (version 7.0.2.11).
3
ESAT-6 Oligomerization Analysis by SEC-MALS
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SEC-MALS was performed on an AKTApure FPLC (Cytiva) with a DAWN MALS detector (Wyatt). A total of 100 µg of ESAT-6 was buffer exchanged with a Zeba spin column immediately prior to each experiment and injected onto a Superdex 75 increase 10/300 column at a flow rate of 0.5 mL/min at 4 °C. The mobile phase was 10 mM Citrate, 300 mM NaCl, pH 4.5 or pH 7.5. Buffers were sterile filtered to 0.1 µm and degassed prior to use. Data analysis was performed using ASTRA 8.1.2.1 (Wyatt).
4
SEC-MALS Analysis of Protein Samples
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All protein samples were centrifuged at 9,500×g for 5 min at 4°C immediately before injecting into a 100 μL sample loop. SEC-MALS was performed using a 16 angle Dawn Heleos II light-scattering instrument and Optilab TrEX differential refractometer (Wyatt Technologies) connected in-line with a Superdex 200 Increase 10/300 GL column (Cytiva), controlled by an AKTA Pure FPLC (Cytiva) housed at ∼10°C. Molecular weight was calculated using the ASTRA software (v7.1.4, Wyatt) based on the Zimm plot analysis and using a protein refractive index increment, dn dc−1 = 0.185 L g−1. Proteins from elution factions were visualized using 15% (w/v) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and Coomassie blue staining. All experiments performed in the presence of divalent cations used 5 mM CaCl2 or 5 mM MgCl2.
5
Purification of Anti-RBD IgGs and hFc-ACE2 Fusions
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Anti-RBD IgGs and hFc-Ace2
fusions were purified using a 5 mL MAbSelect SuRe Prism column on
the AKTA Pure FPLC (Cytiva). Filtered cell supernatants were diluted
with 1/10 volume of 10× PBS. The AKTA system was equilibrated
with 1× PBS for A1, 100 mM glycine pH 2.8 for A2, 0.5 M NaOH
for B1, 1× PBS for the buffer line, and H2O for the
sample lines. The protocol involved washing the column with A1, then
loading the sample in Sample line 1 until air was detected in the
air sensor of the sample pumps, followed by five column volume washes
with A1, and elution of the sample by flowing of 20 mL of A2 (directly
into a 50 mL conical containing 2 mL of 1 M Tris pH 8.0) followed
by five column volumes of A1, B1, and A1. The resultant Fc-containing
samples were concentrated using 50 or 100 kDa cutoff centrifugal concentrators
(Amicon). Proteins were buffer exchanged using a PD-10 column (Sephadex)
that had been preequilibrated into 20 mM HEPES and 150 mM NaCl. IgG-ACE2
fusions were further purified using the S6 column on the AKTA as described
above.
fusions were purified using a 5 mL MAbSelect SuRe Prism column on
the AKTA Pure FPLC (Cytiva). Filtered cell supernatants were diluted
with 1/10 volume of 10× PBS. The AKTA system was equilibrated
with 1× PBS for A1, 100 mM glycine pH 2.8 for A2, 0.5 M NaOH
for B1, 1× PBS for the buffer line, and H2O for the
sample lines. The protocol involved washing the column with A1, then
loading the sample in Sample line 1 until air was detected in the
air sensor of the sample pumps, followed by five column volume washes
with A1, and elution of the sample by flowing of 20 mL of A2 (directly
into a 50 mL conical containing 2 mL of 1 M Tris pH 8.0) followed
by five column volumes of A1, B1, and A1. The resultant Fc-containing
samples were concentrated using 50 or 100 kDa cutoff centrifugal concentrators
(Amicon). Proteins were buffer exchanged using a PD-10 column (Sephadex)
that had been preequilibrated into 20 mM HEPES and 150 mM NaCl. IgG-ACE2
fusions were further purified using the S6 column on the AKTA as described
above.
6
Transient ABBV-022 Expression in CHO Cells
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ABBV-022 was transiently expressed in CHO 3E7 cells for 7 days following transfection. Anti-TNC D IgG production is similar to previously described [10, 18] . The monoclonal antibody targeting TNC D and ABBV-022 were puri ed via protein A through mediated capture of the Fc and VH3 domains of the IgG and scFv, respectively. For each TNC D targeting protein, the clari ed supernatant was puri ed on a AKTAPure FPLC (Cytiva, Marlborough, MA) via ProteinA a nity capture using a HiTrap MabSelect SuRe column (Cytiva, Marlborough, MA) and eluted at low pH. A second puri cation was performed using a Superdex 200 pg (Cytiva, Marlborough, MA) size exclusion column using tris buffered saline to yield >95% protein purity as determined by analytical liquid chromatography (Agilent, Santa Clara, CA).
Endotoxin level were quanti ed to be <1 EU/mg (Charles River Laboratories, Wilmington, MA).
Endotoxin level were quanti ed to be <1 EU/mg (Charles River Laboratories, Wilmington, MA).
7
Oligomeric State Determination of McpZ
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