SEC-MALS was performed using a Superdex 200 Increase 10/300 Gl column (Cytiva) connected to an AKTA pure FPLC system (Cytiva). A DAWN HELEOS II detector (Wyatt) and an Optilab TrEX differential refractometer (Wyatt) were used to estimate the molecular weight of MRS258–333 under various experimental conditions. The entire in-line FPLC/MALS system was housed in cold cabinet maintained at ∼10°C. Data were obtained for four different protein concentrations: 0.45 mg/ml, 0.90 mg/ml, 2.5 mg/ml, and 5 mg/ml in 20 mM Tris (pH 8), 150 mM NaCl, and 1 mM DTT, using 100 µl injections of sample at each concentration. MALS molecular weights were determined in the accompanying ASTRA software (version 7.1.4; Wyatt) using Zimm plot analysis and a protein refractive index increment (dn/dc) = 0.185 L/g. Divalent cation containing experiments were performed by supplementing the running buffers and protein samples with 5 or 10 mM MgCl2 and CaCl2, as indicated.
Akta pure fplc system
Manufactured by Cytiva
The AKTA pure FPLC system is a fast protein liquid chromatography (FPLC) instrument designed for high-performance protein purification. It is a flexible and automated system that supports a wide range of chromatographic techniques, including ion exchange, size exclusion, and affinity chromatography. The AKTA pure FPLC system is capable of performing complex purification protocols with high resolution and reproducibility.
Automatically generated - may contain errors
Lab products found in correlation
Akta pure, by Cytiva (7 mentions)
Superdex 200 10 300 gl, by Cytiva (2 mentions)
Superdex 200 increase 10 300 gl column, by Cytiva (1 mentions)
Dawn heleos 2 detector, by Wyatt Technology (1 mentions)
Astra software, by Wyatt Technology (1 mentions)
Optilab t rex differential refractometer, by Wyatt Technology (1 mentions)
Hitrap sp hp column, by Cytiva (1 mentions)
Hitrap, by Cytiva (1 mentions)
Synergy mx, by Agilent Technologies (1 mentions)
Pierce bca, by Thermo Fisher Scientific (1 mentions)
Sw41ti, by Beckman Coulter (1 mentions)
Sw32ti, by Beckman Coulter (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
Expicho s cells, by Thermo Fisher Scientific (1 mentions)
Q sepharose fast flow column, by Cytiva (1 mentions)
Q sepharose high performance column, by Cytiva (1 mentions)
8 protocols using akta pure fplc system
1
SEC-MALS Analysis of MRS2 Protein
2
Recombinant Expression and Purification of MCUb
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The nucleotide sequence encoding the NTD of H. sapiens MCUb (GenBank: NP_060388.2) was cloned into a pET-28a vector using the NheI and XhoI restriction sites. Transformed BL21(DE3) codon plus E. coli cells were grown in Luria-Bertani (LB) medium containing kanamycin (60 μg mL−1) at 37°C until the optical density (OD) at 600 nm reached ∼0.6–0.8. Subsequently, 0.35 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce 6×His-MCUb58-159 expression over 16 h at 24.5°C. Harvested cells were lysed by sonication in 20 mM Tris (pH 8.5), 150 mM NaCl, 1 mM DTT. Purification was performed as described in the nickel-nitrilotriacetic acid (Ni2+-NTA) agarose beads manufacturer protocol (HisPur, Thermo Fisher). The 6×His tags were removed by overnight incubation with ∼1 Unit of bovine thrombin (EMD millipore) per mg of protein. Size-exclusion chromatography (SEC) through a Superdex 200 10/300 GL (Cytiva), connected to an AKTA pure FPLC system (Cytiva) at 10°C, was performed as the final purification step in 20 mM Tris (pH 8.5), 150 mM NaCl, 1 mM DTT. The protein concentration of MCUb58-159 was estimated using an extinction coefficient (280 nm) of 0.4280 (mg/mL)−1 cm−1.
3
FAM210A-dMTS Purification via Cation Exchange
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A HiTrap HP SP column (Cytiva, Cat. # 17115201) and an AKTA pure FPLC system (Cytiva) were used to perform cation exchange chromatography to further purify FAM210A-dMTS. The column was initially equilibrated in 50 mM Tris-Cl pH 7.5, 5% glycerol, 1 mM DTT, 0.01% DDM, and 50 mM NaCl. FAM210A-dMTS was buffer exchanged into the same buffer immediately before the chromatography run using a 10 kDa MWCO centrifugal filter. A 50 mM-1M NaCl gradient in the same buffer was used to elute FAM210A-dMTS. Fractions were collected for further concentration and analysis.
4
His-tagged Protein Purification Protocol
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His-tagged recombinant proteins were expressed in Rosetta2 (DE3) pLysS bacteria (Novagen) and purified on immobilized metal chelate affinity chromatography columns (Bio-Rad) using an AktaPure FPLC system (Cytiva). Purity was assessed by SDS-PAGE.
5
Isolation and Characterization of Human HDL
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DGUC was completed, as previously reported (71 ). Native human HDL (1.063–1.021 g/L) were isolated by sequential DGUC using an Optima XPN-80 Ultracentrifuge with SW32Ti or SW41Ti rotors (Beckman-Coulter). Lipoproteins were dialyzed in 1× PBS (4L) for at least four bucket changes and concentrated with 3 kDa m.w. cutoff filters (Millipore). Isolated human DGUC-HDL, cell culture media, or plasma samples were injected into an AKTA pure FPLC system (Cytiva) with three tandem Superdex-200 Increase SEC columns (Cytiva) and collected in 1.5 ml fractions in running buffer (10 mM Tris-HCl, 0.15 M NaCl, 0.2% NaN3), as previously described(21). SEC fraction fluorescence was measured with microplate fluorometer (SynergyMx, Biotek). DGUC-HDL or individual SEC fractions were assessed for total protein (Pierce BCA, ThermoFisher), TC, PC, and triglyceride (Pointe Scientific) by colorimetric assays.
6
Antibody Production and Purification
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Plasmids encoding the heavy and light chains of mAbs were prepared using the PureYield Plasmid Midi- or Maxi-prep system and filter sterilized using a 0.22-μm filter. Plasmids were then transfected into ExpiCHO-S cells (Thermo Fisher Scientific) as per the manufacturer’s instructions. Culture supernatant was harvested via centrifugation at 7 days after transfection and antibodies were purified using Protein A affinity chromatography on an AKTA pure FPLC system (Cytiva) as per the manufacturer’s instructions. Purified antibodies were concentrated and buffer-exchanged into phosphate-buffered saline (PBS) using a 30-kDa molecular weight cutoff Amicon Ultra Centrifugal Filter (Merk) and stored at −20°C until use.
7
Purification and Refolding of MCU72-189
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The pET-28a MCU72-189 vector and associated D147R mutant were from the previously described work.33 (link) WT or D147R MCU72-189 constructs were transformed in BL21 (DE3) codon plus E. coli. Transformed cells were grown in LB medium containing kanamycin (60 μg mL−1) at 37°C to an OD 600 nm of ∼0.6–0.8, and 200 μM of IPTG was added to induce expression over a 16 h period. The harvested cells were purified under denaturing conditions using (Ni-NTA) resin (HisPur) according to the manufacturer instructions. The protein was refolded in 20 mM Tris, 150 mM NaCl and 1 mM DTT, pH 8.8 by dialysis. After overnight thrombin cleavage (∼1 U/mg protein), the protein was further purified by SEC through a Superdex 200 10/300 GL (Cytiva) connected to an AKTA pure FPLC system (Cytiva) at 10°C. This final purification step buffer was 20 mM Tris (pH 8.5), 150 mM NaCl, 1 mM DTT. The protein concentration of MCU72-189 was estimated using an extinction coefficient (280 nm) of 0.3693 (mg/mL)−1 cm−1.
8
Delipidation and Purification of ApoA-I
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DGUC-HDLs were delipidated as previously described (9 (link)). Briefly, 6 ml of ice-cold methanol:chloroform (1:1) was added to 1 ml of lyophilized DGUC-HDL, mixed, and incubated on ice for 30 min. Chilled methanol (4 ml) was added to the mixture, centrifuged (3000 rpm) for 10 min, removed supernatants, and repeated until pellet turned white. Chilled methanol (10 ml) was re-added, mixed, and recentrifuged. The pellet was dried under nitrogen, resolubilized with 6M guanidine HCl, and dialyzed in ammonium bicarbonate (10 mM) overnight. ApoA-I was isolated from delipidated HDL with 6M Urea by anion exchange chromatography with Q-Sepharose Fast Flow column coupled with a Q-Sepharose High-Performance column on an AKTA pure FPLC system (Cytiva). ApoA-I fractions were confirmed using Coomassie-based Aquastain (BulldogBio) on an SDS-PAGE gel and were dialyzed in ammonium bicarbonate (10 mM).
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