PVX-A647 nanoparticle preparations were analyzed by SEC/FPLC using a Superose6 column on the ÄKTA Explorer chromatography system (GE Healthcare). Samples (100 µL of 1 mg mL−1) were analyzed at a flow rate of 0.5 mL min−1, using 0.1 M potassium phosphate buffer (pH 7.4).
Kta explorer chromatography system
Manufactured by GE Healthcare
Sourced in United Kingdom, United States, Germany, Switzerland, Sweden
The ÄKTA Explorer chromatography system is a versatile and automated liquid chromatography platform designed for a wide range of applications in purification and analytical processes. The system features accurate flow control, precise gradient formation, and robust detection capabilities to facilitate efficient separation and analysis of biomolecules.
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Lab products found in correlation
Mut express 2 fast mutagenesis kit v2, by Vazyme (1 mentions)
Plhcx flag mpkm2, by Addgene (1 mentions)
Histrap hp column, by GE Healthcare (1 mentions)
Pmal c5x, by New England Biolabs (1 mentions)
Coomassie plus protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Sulfo cy5 nhs, by Lumiprobe (1 mentions)
Hiload superdex 200 pg column, by GE Healthcare (1 mentions)
Dylight650 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Ni nta superflow cartridge, by Qiagen (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Hitrap protein a hp, by GE Healthcare (1 mentions)
Hitrap, by Cytiva (1 mentions)
9 protocols using kta explorer chromatography system
1
Characterizing PVX-A647 Nanoparticles by SEC/FPLC
2
Cloning and Purification of PKM1 and PKM2
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The full length cDNAs encoding PKM1 and PKM2 were cloned from pLHCX-Flag-mPKM1 (Plasmid #42511, Addgene) and pLHCX-Flag-mPKM2 (Plasmid #42512, Addgene) by PCR technique using forward primer: 5'- GTACGAATTCATGCCGAAGCCACACAGT-3'; reverse primer: 5'- GTACCTCGAGTCAAGGTACAGGCACTACAC-3'. Following cleavage with restriction enzymes XhoI and EcoRI, the PCR products were respectively ligated into bacterial expression vector pET-28a with T4 DNA ligase. For the generation of mutated PKM2 (C31S) plasmid, the point mutation was achieved using Mut Express II Fast Mutagenesis Kit V2 (Nanjing Vazyme Biotech Co., Ltd) with forward primer 5′-GGACATGTGTTCCAGGAAGGTGTCAGCCATGG-3′ and reverse primer 5′-TCCTGGAACACATGTCCCGCCTGGACATTGACTCTG-3′. The construction of expression plasmid was confirmed by DNA sequencing at BGI Hong Kong Company Limited (Hong Kong, China). The cDNA constructs were introduced into E. coli BL21 (DE3) cells while the bacterial colonies were examined for transformation. For protein expressions, the best E. coli BL21 (DE3) transformants were induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30 °C for 6 h. The recombinant proteins were purified with a HisTrapTM HP column under the control of ÄKTAexplorer™ chromatography system from GE Healthcare. The recombinant protein fractions were combined and stored at -80 °C until use.
3
Production and Purification of MBP-Sulf1HD Fusion Protein
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For the investigation of Sulf1HD, a fusion protein of an N-terminal maltose binding protein tag followed by the HD of human Sulf1 (residues K417-K735) (MBP-HD) was produced. Expression of MBP-HD was achieved in Escherichia coli Rosetta 2 (DE3) (Merck, Darmstadt, Germany) using a plasmid that is based on pMAL-c5X (New England Biolabs, Frankfurt, Germany) and which has been already described27 (link). The protein was purified following an established protocol22 (link). Briefly, after expression, harvested cells were resuspended in phosphate buffered saline (PBS, prepared from substances of analytical grade (purity ≥99%) and sterile filtered, pH7.3) on ice and lysed. Cleared supernatant was loaded on an amylose affinitiy column (MBP-Trap HP 5 GE Healthcare, Little Chalfont, UK) using an ÄKTAexplorer chromatography system (GE-Healthcare) at 4 °C. After washing out unbound protein with PBS, pH7.3, bound proteins were eluted in one step to 100% elution buffer (10 mM D-maltose in PBS, pH7.3, Sigma Aldrich, purity ≥95%). Fractions of 2 ml each were collected and analyzed via Bradford assay (Coomassie Plus Protein Assay Reagent, Thermo Fisher Scientific), SDS-Polyacrylamide electrophoresis and subsequent Coomassie Brilliant Blue staining22 (link).
4
Purification and Characterization of Chloroplast F1Fo ATP Synthase
5
Fluorescent Labeling of Plant Virus Nanoparticles
6
Chromatographic Purification of CPMV
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200 μL of native and inactivated CPMV samples were eluted through a Superose 6 increase column using the ÄKTA Explorer chromatography system (GE Healthcare). The flow rate was set to 0.5 mL min−1 in 0.1 M KP buffer (pH 7.0) and the absorbance at 260 nm and 280 nm was recorded.
7
Purification of Photoactivatable PhyB Tetramers
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The production of PhyB1-651-AviTag-His6 was performed as described before [43 (link)]. Briefly, PhyB1-651-AviTag-His6 was expressed in E. coli BL21(DE3) cells and purified from bacterial lysates by affinity chromatography on a Ni-NTA Superflow cartridge (Qiagen, Hilden, Germany) using an Äkta Explorer chromatography system (GE Healthcare, Freiburg, Germany). Subsequently, PhyB tetramers (PhyBt) were formed and purified as described previously [44 (link)]. Briefly, Ni-NTA column-purified PhyB1-651-AviTag-His6 was mixed in a 10:1 molar ratio with DyLight650-conjugated streptavidin (Thermo Fisher Scientific, Darmstadt, Germany) and the mixture was incubated for 2 h at room temperature in the dark. Then, PhyBt were separated from the excess of PhyB monomers by size-exclusion chromatography on a HiLoad Superdex 200 pg column (GE Healthcare, Freiburg, Germany) using PBS with 0.5 mM TCEP as mobile phase. The eluate fractions containing the purified tetramers were pooled and concentrated using Spin-X UF 10k (Corning, Kaiserslautern, Germany) centrifuge concentrators to a final concentration between 0.5 and 2 µM.
8
Antibody Fragment Generation and Characterization
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Example 6
Generation of Antibody Fragments
The F(ab′))2 fragment and the Fc-region fragment were prepared by cleavage of the full-length antibody 1:1 diluted with 100 mM Tris, pH 8.0, by adding 1 μg IdeS cysteine protease per 50 μg antibody and incubation for 2 h at 37° C. The resulting cleavage products F(ab′)2 and Fc were separated on a size exclusion chromatography (SEC) column (Superdex 200, GE Healthcare, Zurich, Switzerland) using an ÄKTA Explorer chromatography system (GE Healthcare, Uppsala, Sweden) and the peak fractions were pooled. Molecular weight standards on the same column served to identify the two cleavage products based on their retention times.
Retention times of full-length antibodies varied notably. In contrast, the retention times of the respective Fc portions of all tested antibodies virtually did not differ from each other (<1%).
When plasmin was used for cleavage of the full-length antibodies, the same findings were obtained (data not shown).
9
Chromatographic Purification of IgG
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All chromatography runs were performed on an ÄKTA explorer chromatography system (GE HealthCare, Uppsala, Sweden). UV (280 nm, 260 nm), pH, conductimetry (mS) and flow rate were monitored during each run. For the EPO-POROS columns, the tests were performed with either a 1.4-fold dilution of IVIg (to a concentration of IgG of 35.7 mg/ml) or undiluted plasma. A flow rate of 0.5 ml/min (0.5 column volume (CV)/min) was applied. For the NHS-Sepharose columns, various volumes of diluted IVIg (35.7 mg/ml) or undiluted plasma samples were used as indicated in the results section. Different flow rates ranging from 0.5 ml/min (0.1 CV/min) to 0.1 ml/min (0.02 CV/min) were initially tested and a rate of 0.1 ml/min (0.02 CV/min) was then selected. For the HiTrap Protein A HP (5 ml, GE HealthCare, Uppsala, Sweden) different volumes (1 ml, 2 ml, 3 ml and 5 ml) of either IVIg (35.7 mg/ml) or plasma were used. The buffer system used was PBS (running and washing buffer) for all columns. The volume of wash buffer (PBS) used was 12 ml for all columns. Similarly, elution was performed identically for all columns using 12 ml of 50 mM citrate buffer (pH 3.0). All experimental conditions were tested at least 3 times.
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