From the harvested culture supernatants, HA proteins were purified by a two-step protocol using an ÄKTA Avant 25 system (GE Healthcare Life Sciences). For his-tagged proteins, the material was applied to either a prepacked cOmplete His-tag purification column (Roche) or self-packed HiScale 26/60 column with Ni Sepharose High Performance (GE Healthcare Life Sciences). Following a wash with 1 mM imidazole, the bound proteins were eluted with a step gradient to 300 mM imidazole. For C-tagged protein, the clarified supernatant was loaded on a HiScale 16/20 column (GE Healthcare) packed with an affinity resin that consisted of a C-tag–specific single domain antibody immobilized on Agarose-based beads (Thermo Fisher Scientific). The elution of the C-tagged proteins was performed using a Tris buffer containing 2 M MgCl2. The His-tag– and C-tag–containing elution fractions were pooled and filtered through a Millex-GV 0.22-µM filter membrane (Millipore Sigma). To further polish the purified protein, SEC was performed by running a HiLoad Superdex 200-pg 26/60 column (GE Healthcare Life Sciences). Peak fractions were analyzed on a sodium dodecyl-sulfate polyacrylamide gel electrophoresis, pooled, and, when the concentration was <1 mg/mL, concentrated by centrifugation using Amicon Ultra-15 centrifugal filters with a 10-kDa cutoff.
Kta avant 25 system
Manufactured by GE Healthcare
Sourced in United States, Sweden
The ÄKTA Avant 25 system is a liquid chromatography system designed for protein purification and separation. It is capable of handling flow rates up to 25 mL/min and can be used with a variety of chromatography media. The system is equipped with advanced features for monitoring and controlling the purification process.
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Lab products found in correlation
Superdex 200 hr 10 30 column, by GE Healthcare (2 mentions)
Quick start bradford protein assay, by Bio-Rad (2 mentions)
Complete his tag purification column, by Roche (1 mentions)
Hiscale 16 20 column, by GE Healthcare (1 mentions)
Ni sepharose high performance, by GE Healthcare (1 mentions)
Resource q column, by GE Healthcare (1 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
Gel filtration calibration kit, by GE Healthcare (1 mentions)
Superdex 75 hr 10 30 column, by GE Healthcare (1 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (1 mentions)
9 protocols using kta avant 25 system
1
Purification of His-tagged and C-tagged Proteins
2
Heterologous Expression and Purification of PAH Proteins
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Human PAH proteins were expressed in XL1 blue cells, with or without co-transformation of pGroELS plasmid coding GroEL and GroES bacterial chaperones. The expression was induced by the addition of 100 mM IPTG (Isopropyl-β-D-thiogalactoside) simultaneously with 0.2 mM FAS (ferrous-ammonium-sulfate) to cultivation medium at optical density (OD) = 0.5. PAH proteins were expressed for 18 hours at 37 °C. Cells were collected and sonicated in the buffer comprised of 200 mM NaCl, 20 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), and 0.2 mM Pefabloc, pH = 7.0 at 4 °C. PAH proteins were subsequently purified using the ÄKTA avant 25 system (GE Healthcare Life Sciences Marlborough, Massachusetts, USA) by affinity and size-exclusion chromatography using MBPTrapTM HP 1 mL column (GE Healthcare Life Science) and Superdex 200 HR 10/30 column (GE Healthcare Life Sciences), respectively. For the purification procedure, the column buffer containing 200 mM NaCl, 20 mM HEPES, pH = 7.0 was used. The protein elution was performed using 10 mM maltose admixed with the column buffer. PAH tetrameric forms were collected, if absented the mixture of PAH oligo- and dimers was collected, and spectrophotometrically quantified using Quick StartTM Bradford Protein Assay (Bio-Rad, Hercules, CA, USA).
3
Plasma Protein Separation and Identification
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Four overnight-fasted C57Bl6 mice were anaesthetized using isoflurane and blood was collected via the retro-orbital sinus. Protein from 2 ml collected murine plasma was desalted and rebuffered in 10 mM Tris-HCl, pH 8 using a PD-10 desalting column (GE Healthcare) and further diluted to a final volume of 20 ml using 10 mM Tris-HCl, pH 8. Plasma proteins were separated by anion exchange chromatography using Resource Q column (GE Healthcare, 6 ml) connected to an ÄKTA Avant 25 system (GE Healthcare). After loading, the column was washed with 10 column volumes of binding buffer and bound proteins were eluted by linear salt gradient (0–1 M NaCl). The protein concentration of all fractions was measured and ZAG-containing fractions identified by WB.
4
Purification of Protein Isoforms
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Enriched fractions from a polishing step in the purification process containing mixtures of monomer, monomer variant, and aggregate were dialyzed into water with Spectra/Por regenerated cellulose dialysis tubing (molecular weight cutoff, 12–14 kDa; cat# 25225–281; VWR, Radnor, PA) and concentrated and buffer exchanged into 1× phosphate-buffered saline (cat# 10010023; Thermo Fisher Scientific, Waltham, MA) to ~ 15 mg/mL, using Centriprep 30K centrifugal filters (cat# 4307; Millipore). The mixture was separated with two TSKgel G3000SW columns (21.5 mm × 60 cm, cat# 05147; Sigma-Aldrich) connected in series to an ÄKTA Avant 25 system (GE Healthcare Life Sciences, Little Chalfont, U/K) flowing at 4 mL/min, using 1× phosphate-buffered saline as the running buffer. The eluate was fractionated into 2-mL aliquots and analyzed by analytical high-performance SEC to generate pools of monomer, monomer variant, and aggregate. The pools were further concentrated and buffer exchanged into 20 mM histidine–235 mM sucrose, pH 6.0, using Centriprep 30K centrifugal filters (Sigma-Aldrich) to > 2.5 mg/ml for further analytical characterization.
5
Gel Filtration of DNA-Protein Complexes
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DNA duplex SP (Supplementary Table S1 ) and His-AgeI were used for gel filtration. Gel filtration was carried out at room temperature on an ÄKTA Avant 25 system (GE Healthcare) using a Superdex 75 HR 10/30 column (GE Healthcare) pre-equilibrated with 20 mM Tris–HCl (pH 8.0), 0.2 M NaCl and 5 mM CaCl2. Protein (5–35 μM concentration) and protein–DNA (protein concentration 7 μM, SP DNA (Supplementary Table S1 ) concentration 3.5–140 μM) samples were prepared in 100 μl of the above indicated buffer. Protein elution from the column was monitored by measuring absorbance at 280 nm. The apparent molecular mass was evaluated from the elution volume using a series of standards (Gel filtration Calibration Kit from GE Healthcare).
6
Size-exclusion Chromatography of Recombinant Proteins
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Size-exclusion chromatography was performed at Biocentar d.o.o., Zagreb, Croatia. Recombinant proteins (EsuDRG1-His, EsuDFRP1-His and HsaDRG1-His) were loaded onto size-exclusion Superdex 200 Increase 10/300 GL column (GE Healthcare), pre-equilibrated with 10 mM phosphate buffer, 140 mM NaCl, pH 7.4. Proteins were eluted at 0.5 mL/2 min or 0.5 mL/1 min using an Äkta avant 25 system (GE Healthcare) at 4 °C. Injection volume was 500 µL. The column was calibrated with Bio-Rad Gel filtration standards: Thyroglobulin (670 kDa), γ-globulin (158 kDa), Ovalbumin (44 kDa), Myoglobin (17 kDa), and Vitamin B21 (1.35 kDa).
7
Gel Filtration Purification of Enzyme
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An aliquot (1.0 mL) of the sample obtained from DEAE which presented activity was submitted to Gel filtration. The analysis was performed using Tris-HCl buffer 0.1 M (pH 8) added of 0.15 M NaCl in an ÄKTA Avant 25 System (GE Healthcare, Uppsala, Sweden) on Superdex G 75 (HR10/300GL), PC 3.2/30 column, as described in the manufacturer's instructions. The absorbance of the samples was evaluated at 215 nm and 280 nm. The column was calibrated using a mixture of molecular weight markers (1 mg/mL each): bovine serum albumin, carbonic anhydrase and albumin from chicken egg and a trypsin inhibitor. All the steps were done according to Nascimento et al. (2017) .
8
Antibody Retention Mapping on Resins
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Retention mapping was conducted with mAb-1 for all resins using 1 mL HiTrap columns with a GE Healthcare ÄKTA avant 25 system. Protein was loaded with a 4 min residence time, and protein concentration was monitored through A280 measurements. The feed material (purified antibody) was used as the absorbance at 100% breakthrough.
9
PAH Purification by Chromatography
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Wt PAH and each mutated form were purified by affinity chromatography using the ÄKTA avant 25 system (GE Healthcare Life Sciences), and MBPTrap™ HP 1 ml column (GE Healthcare Life Science) loaded with a column buffer containing 200 mM NaCl and 20 mM HEPES, pH 7.0. Elution of PAH-MBP was performed using 10mM maltose dissolved in the column buffer and proteins were subsequently purified by size-exclusion chromatography using a Superdex 200 HR 10/30 column (GE Healthcare Life Sciences) in the same column buffer. Protein concentration was then spectrophotometrically determined using Quick Start™ Bradford Protein Assay (Bio-Rad).
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