To enable expression of the PGT121-knob fusion proteins, plasmid DNA was amplified using QIAGEN Plasmid Plus Giga Kits and quantified by A260. Individual Expi293F cell cultures, at 3 × 106 cells/mL, per construct, were set up using Expifectamine 293 Transfection kits (Invitrogen), as per the manufacturer’s instructions. Both heavy-chain and light-chain plasmid DNA were co-transfected at 1 mg/L. The cells were cultured for 4 days, centrifuged at 4,000 rpm for 1 hour, and filtered through a 0.22 μm filter. Using an Akta pure (GE Healthcare), Hi-Trap Nickel excel columns (GE Healthcare) were equilibrated with 10 column volumes (CV) of PBS. Cell supernatants were loaded at 1.0 mL/min, and the column was washed with 7× CV of PBS, 0.5 M NaCl. The column was then washed with 7× CV of Buffer A (0.5 M NaCl, 0.02 M imidazole, PBS [pH 7.3]). Protein samples were eluted by isocratic elution with 10× CV of Buffer B (0.5 M NaCl, 0.25 M imidazole, PBS [pH 7.3]), as 1.0 mL fractions. The column was washed with 0.1 M NaOH and re-equilibrated into PBS prior to subsequent loading. Post elution, the protein containing fractions were pooled and buffer exchanged into PBS, using PD-10 columns (GE Healthcare).
Akta pure
Manufactured by GE Healthcare
Sourced in United States, Sweden
AKTA Pure is a versatile liquid chromatography system designed for protein purification. It offers automated control and monitoring of parameters such as flow rate, pressure, and UV absorbance during the purification process. The system is suitable for a wide range of applications, including buffer preparation, sample injection, and fraction collection.
Automatically generated - may contain errors
Lab products found in correlation
Hitrap, by Cytiva (4 mentions)
Histrap ff, by GE Healthcare (3 mentions)
Superose 12 10 300 gl column, by GE Healthcare (2 mentions)
Amicon centrifugal filter, by Merck Group (2 mentions)
Gene pulser xcell electroporation system, by Bio-Rad (2 mentions)
Superdex s75 10 60 column, by GE Healthcare (2 mentions)
Escherichia coli t7 express plac 1 y cells, by New England Biolabs (2 mentions)
Complete edta free, by Roche (2 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (2 mentions)
Vivaspin 20 ultrafiltration unit, by Sartorius (2 mentions)
Nico21 de3, by New England Biolabs (2 mentions)
Optilab rex, by Wyatt Technology (2 mentions)
Expifectamine 293 transfection kit, by Thermo Fisher Scientific (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
Plasmid plus giga kit, by Qiagen (1 mentions)
Prescission protease, by GE Healthcare (1 mentions)
Superose 6 increase 10 300 column, by GE Healthcare (1 mentions)
Capto, by Cytiva (1 mentions)
Hitrap q ff column, by GE Healthcare (1 mentions)
200 increase 10 300 gl column, by GE Healthcare (1 mentions)
64 protocols using akta pure
1
PGT121-knob Fusion Protein Expression and Purification
2
Molecular Weight Distribution of Fish Protein Hydrolysate
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Molecular weight (MW) distribution of FPH was determined by gel filtration chromatography using an FPLC system (AKTA pure, GE Healthcare Life Sciences, Chicago, IL, USA) coupled with two gel filtration columns: Superdex 200 increase10/300 GL and Superdex peptide, 10/300 GL. The eluent used was 0.025 M phosphate buffer (pH 7) containing 0.2 g/L of sodium azide and 8% NaCl at a flow rate of 0.5 mL/min. Elution was monitored at 280 nm and Thyroglobulin (669 kDa), Ferritin (440 kDa), Aldolase (158 kDa), Conalbumin (75 kDa), Ovalbumin (44 kDa), Carbonic anhydrase (29 kDa), Ribonuclease A (14 kDa) and Whey peptide (1 kDa) were used to perform the molecular weight standard curve. The results were expressed in milli Absorbance Units (mAU) per eluted volume (mL) [43 (link)].
3
Cj2Cas9 Protein Expression and Purification
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Full-length Cj2Cas9 cDNA was sub-cloned into the bacterial expression vector pGEX-6P-1. Cj2Cas9 protein was expressed in E. coli C43 (DE3) cells and purified as previously described previously (42 (link)). Cj2Cas9 expression was induced by 0.3 mM IPTG at 16°C. After overnight induction, cells were collected by centrifugation. Cj2Cas9 was resuspended in buffer (25 mM Tris–HCl, pH 8.0, 1 M NaCl, 3 mM DTT). Cells were disrupted by sonication and cell debris was removed by centrifugation. The lysate was purified using glutathione sepharose 4B (GS4B) beads. The bound proteins were cleaved with precision protease overnight at 4°C to remove the GST tag. The cleaved Cj2Cas9 protein was eluted from the GS4B resin and further fractionated by heparin sepharose column and ion exchange chromatography via FPLC (AKTA Pure, GE Healthcare).
4
Affinity Purification of GST Fusion Proteins
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Enzyme purification was performed according to the methods described in previous reports [52 (link),53 (link)] with modifications. Cell cultures (1 L) were separated via centrifugation at 8000× g for 10 min. The cell pellets were resuspended in 40 mL binding buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.0) and subjected to 400 pulses of sonication (400 W, 3 s each with a 5 s interval) in an ice-water bath [54 (link)]. Following centrifugation (13,000× g) at 4 °C for 30 min, the supernatant was passed through a 0.22 μm filter and applied to a GSTrap (GE Healthcare, Marlborough, MA, USA) FF column of the liquid chromatography system, AKTAPure (GE Healthcare, Marlborough, MA, USA), and GST fusion proteins were eluted via a GSTrap FF column using 10 mM reduced glutathione according to standard affinity chromatography. GST fusion proteins were digested using PreScission protease (GE Healthcare, Marlborough, MA, USA) according to the manufacturer’s instructions.
5
PAPP-A Dimerization Mechanism Analysis
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For the SEC assay to examine dimerization mechanism, purified WT PAPP-A, PAPP-A (E483A), PAPP-A2 WT, PAPP-A monomeric mutants, and C-terminal truncation constructs (PAPP-A (1132) and PAPP-A (1267)) were thawed and centrifuged at 15,000 g for 5 min at 4 °C to remove any potential precipitates. Concentration of the PAPP-A proteins were normalized to 1.4 μM, then 0.2 mL of each protein was injected onto a Superose 6 Increase 10/300 column (Cytiva 29-0915-96) connected to an AKTA Pure (GE Healthcare). The system was run at 0.5 mL/min for 1 h using 1X PBS as the mobile phase. UV280 measurements were obtained directly from the instrument. The fractions from retention volume between 11.5-16.5 ml were run on a 4-12% Bis-Tris SDS-PAGE, stained with Coomassie Protein Stain (InstantBlue® ab119211) and destained with Milli Q Water.
6
Liver Protein Fractionation by SEC
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Liver was lysed with TNE buffer. Lysates (0.5 ml, 3.5 mg/ml) were applied to a Superose 6 10/300 GL column using an AKTA Pure (GE Healthcare). The flow rate was 0.5 ml/min and 0.5 ml fractions were collected.
7
Size Exclusion Chromatography of Proteins
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Size exclusion chromatography was performed using AktaPure and a Superose 12 10/300 GL column (GE Healthcare, Inc). A buffer consisting of 10 mM HEPES pH 8.0, 100 mM NaCl, and 10 mM MgCl2 was used to run ≈10–20 μM (0.2–0.5 mg/mL) protein over the column at 0.1–0.25 mL/min. Additional 1 mM DTT was used for proteins containing cysteines. For gel filtration with ligands, 500 μM PRPP and 100 μM 9-deazaguanine were included in the mobile phase buffer where necessary. A gel filtration standard (Bio-Rad) was used to establish molecular weight, and bovine serum albumin (BSA) was included as an additional marker.
8
Recombinant Ferritin-like Protein Purification
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The crude lysate supernatant was mixed with the optimized buffer (200 mM NaAc/AcH, 2.0 M NaCl pH 4.4) in a ratio of 1:1 and divided into 15‐mL centrifuge tubes, each filled with 10 mL. The tubes were placed in a water bath at 75℃ for 10 min and chilled in ice water. The mixture was subsequently clarified by centrifugation at 10,000 rpm for 30 min and the supernatant was discreetly drawn out. The solution conductivity was modulated to about 160 mS/cm by 4 M (NH4)2SO4 and the solution pH was adjusted to pH 6.5 by 1.0 M Tris. The clarified supernatant then proceeded to purification system (AKTA pure, GE healthcare) and a pre‐packed Capto Butyl column was implemented and equilibrated with buffer A (10 mM PB, 1.2 M (NH4)2SO4 pH 6.5). After loading sample, the column was washed with 30% buffer B (10 mM PB pH 6.5) until the UV280 signal baseline tended to be stable. Eventually, the protein was gradually eluted by 90% buffer B. All the intermediate samples were collected during the whole process and eventually analyzed by SDS‐PAGE (13.5%). The resulted rhFTL was dialyzed to PBS buffer and finally stored at 4℃.
9
Protein Purification via Strong Anion Exchange
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Supernatants from the optimal precipitation step were pooled, exchanged by diafiltration into 100 mM pH 2.73 citrate buffer containing 50 mM ammonium sulfate (sample buffer) and stored at 4°C prior to chromatography. Strong anion exchange FPLC was performed in flowthrough mode using a 5 mL HiTrap§ Q FF column on an AKTA Pure instrument (GE Healthcare Life Sciences, Marlborough, MA). The flow path and column were equilibrated with 8 column volumes of sample buffer, followed by manual washing and equilibration of the 500 μL sample loop, injection of 1000 μL of sample at a flow rate of 10 mL/min, and a further wash with sample buffer at a flow rate of 10 mL/min. A single peak was observed to flow through immediately and was collected for analysis.
10
Purification and Analysis of Lutein Nanoparticles
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Lutein ND samples were centrifuged at 15,000 × g for 10 min immediately prior to gel permeation chromatography on a Superdex 200 Increase 10/300 GL column fitted to a GE AKTA Pure fast protein liquid chromatography (FPLC) instrument. Four hundred μl aliquots of lutein ND were applied to the column. Samples were eluted with PBS at a flow rate of 0.75 ml/min. Absorbance was continuously monitored at 280 nm, with collection of 2.0 ml fractions. Fractions corresponding to absorbance peaks were pooled and concentrated to ~500 μl by centrifugal filtration (3,000 Da MWCO). The 280 nm absorbance peaks eluting between 8.2 to 10.2 ml and 12.2–14.2 ml, respectively, were analyzed for phospholipid, lutein, and apoA-I content. EYPC content was determined using a LabAssay Phospholipid kit (Wako Pure Chemical Corp., Japan) according to the manufacturer’s instructions. Lutein content was measured spectroscopically and apoA-I content was determined using the bicinchoninic acid (BCA) assay (Thermo-Fisher) with bovine serum albumin as standard.
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