The cell pellet was resuspended in lysis buffer (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 5% v/v glycerol, 0.3% v/v Triton X-100, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride) and broken via French pressure cell press. The cell lysate was centrifuged at 12,000× g for 30 min at 4 °C to remove insoluble debris. The supernatant was filtered through a 0.45 μm filter and passed through a glutathione S-transferase (GST) Sepharose affinity column (GE Healthcare Life Sciences, Beijing, China) equilibrated with 10 mM Tris-HCl pH 8.0. The target protein was eluted with 10 mM Tris-HCl pH 8.0 containing 10 mM reduced glutathione. The purified fusion protein was digested overnight at 4 °C with TEV protease (Sangon Biotech, Shanghai, China) and then passed through a GST Sepharose affinity column to remove the GST tag. The recombinant protein was then loaded onto a MonoQ 5/50 GL anion exchange column (GE Healthcare Life Sciences, Beijing, China) and eluted using a linear NaCl gradient. The last step of purification was conducted using a Superdex 200 10/300 GL column (GE Healthcare Life Sciences, Beijing, China) on ÄKTAprime plus system (GE Healthcare Life Sciences, Beijing, China). The gel filtration fractions were checked by SDS-PAGE, and pure fractions were pooled and concentrated to 10 mg/mL for crystallization experiments.
Ktaprime plus system
Manufactured by GE Healthcare
Sourced in United States, Sweden
The ÄKTAprime plus system is a compact, automated chromatography system designed for protein purification and analysis. It features a programmable workflow, automated liquid handling, and intuitive software for efficient and reproducible purification of biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Hitrap, by Cytiva (5 mentions)
Histrap hp column, by GE Healthcare (3 mentions)
Histrap hp 5 ml column, by GE Healthcare (2 mentions)
Gene pulser apparatus, by Bio-Rad (2 mentions)
Image studio v3, by LI COR (2 mentions)
Imac sepharose 6 fast flow, by GE Healthcare (2 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
Monoq 5 50 gl anion exchange column, by GE Healthcare (1 mentions)
Pei max, by Polysciences (1 mentions)
Isopropyl β d thiogalactopyranosid iptg, by Carl Roth (1 mentions)
T7 primer, by Macrogen (1 mentions)
Micropulser electroporation apparatus, by Bio-Rad (1 mentions)
E coli bl21 star, by Thermo Fisher Scientific (1 mentions)
Hitrap desalting column, by GE Healthcare (1 mentions)
Gstrap hp column, by GE Healthcare (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Prescission protease, by GE Healthcare (1 mentions)
Superose 12 10 300 gl column, by GE Healthcare (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Coomassie protein assay, by Thermo Fisher Scientific (1 mentions)
35 protocols using ktaprime plus system
1
Purification of Recombinant Protein
2
Recombinant Antibody Purification using Protein A
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purification of the recombinant antibodies was conducted on a ÄKTAprime plus system (GE Healthcare Life Sciences) at 4°C using a 1 mL HiTrap® Protein A HP pre-packed Protein A Sepharose® column (Cytiva). Briefly, the column was first equilibrated in buffer A (20 mM sodium phosphate, 150mM NaCl, pH 7.5) before the sample was loaded on the column. The flow rate for all steps was 1 mL/min. The column was washed with buffer A and buffer B (20 mM Sodium Phosphate, 500 mM NaCl, pH 7.5). The recombinant antibodies were eluted from the column with 100 mM sodium acetate, pH 3.5 and the collected fractions were immediately neutralized with 1M Tris, pH 9. The fractions were pooled and dialyzed against sterile-filtered PBS. The ÄKTAprime plus system and all glasswork was treated with 30% hydrogen peroxide (VWR) to reduce the presence of endotoxin.
3
Purification of Recombinant NSP1 and NSP11
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Histidine‐tagged, recombinant NSP1 and NSP11 were purified in a HisTrap HP affinity column (GE Healthcare Life Sciences, Chicago, IL) by affinity chromatography with an ÄKTAprime Plus system (GE Healthcare Life Sciences), following the manufacturer's protocol. The Lowry method was chosen to quantify eluted proteins.
4
Afucosylated Anti-SARS-CoV-2 Spike IgG1 Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expression vector (pcDNA3.1) encoding human monoclonal IgG1 specific for spike protein of SARS-CoV-2 (COVA1–18, described by Brouwer and co-workers40 (link)) was transfected in HEK 293 Freestyle cells using PEI MAX (Polysciences) as previously reported.41 (link) To enrich for afucosylated IgG1, 0.2 mM 2-deoxy-2-fluoro-l-fucose (Carbosynth) was added to the culture 1 h prior to transfection. After 5 days, the supernatant was harvested, filtered, and antibodies were purified on an ÄKTAprime plus system (GE Healthcare Life Sciences) by affinity chromatography using a protein A HiTrap HP column (GE Healthcare Life Sciences), as previously described.18 (link) Similar procedure was applied for production of low and high fucosylated anti-Rhesus D (anti-RhD) IgG1. Briefly, variable regions for anti-RhD IgG1 were cloned into a pEE6.4 expression vector containing coding sequence for an anti-RhD heavy chain and produced as recently reported.42 (link)
5
Purification of PCMV-R2 Glycoprotein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The R2 fragment of the gB of PCMV/PRV was purified as described14 (link). In brief, 500 mL E. coli BL21 cells containing the pET16b expression vector encoding PCMV-R2 were induced with 1 mM isopropyl-β-D-thiogalactopyranosid (IPTG) (Roth, Karlsruhe, Germany) at an OD600 of 0.7 and cultivated at 37 °C for 2 h. Cells were harvested, centrifuged, resupended in PBS and centrifuged again. 2 g wet cells were dissolved in 10 mL 8 M urea, 0.5 M NaCl, 15 mM imidazole, 20 mM Tris pH 7.5. After a 20 min spin at 50,000 × g the supernatant is applied to a 1 mL HisTrap HP column installed on a Äkta Prime Plus system (both GE Healthcare, Chicago, Illinois, USA) using the HisTrap affinity application template with the following buffers: (1) 6 M urea, 0.5 M NaCl, 15 mM imidazole, 20 mM Tris pH 7.5; (2) 6 M urea, 0.5 M NaCl, 500 mM imidazole, 20 mM Tris pH 7.5. PCMV-R2 eluted in two peaks as was shown by polyacrylamide-gel electrophoresis. Both fractions were used for PCMV antibody detection.
6
Recombinant Expression and Purification of AMA-1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein representing amino acids 1 to 546 of AMA-1 was expressed in Escherichia coli, as previously described [10 (link), 24 (link)]. Briefly, the recombinant plasmid containing pUC57/AMA-1 synthetic gene (GenScript) was resuspended and transformed with competent cells from Escherichia coli XL-1 Blue (Phoneutria, Brazil). After positive clones were confirmed by digestion with the enzymes XhoI and NheI, the AMA-1 insert was subcloned into a bacterial expression vector pET28aTEV. Electrocompetent E. coli BL21-Star (Thermo Fisher Scientific, USA) cells were transformed with the recombinant plasmid pET28a-TEV/AMA-1 by electroporation using a MicroPulser Electroporation Apparatus (Bio-Rad Laboratories, USA). Correct gene insertion was confirmed by colony PCR and using T7 primers (Macrogen, South Korea). Protein large scale expression was obtained after the addition of 1mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubation for 3 h at 37°C at 180rpm. Cells were ruptured by a high-pressure homogenizer and soluble fractions were obtained by centrifugation. The recombinant protein was purified using Ni2+ affinity chromatography with HisTrap HP 5 mL column (GE Healthcare, USA) coupled to an ÄKTA Prime Plus system (GE Healthcare, USA). The purified AMA-1 protein, having 546 amino acids and predicted molecular weight of 62kDa, was separated by SDS-PAGE.
7
Anaerobic Purification of Glutathione S-Transferase
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Asp1 purifications were carried out under strictly anaerobic conditions at RT. Lysate was applied to a GSTrap HP column (GE Healthcare, Little Chalfont, UK) with a bed volume of 5 ml equilibrated with 25 mM Tris–HCl pH 8.0 and 150 mM NaCl using an ÄKTAprime plus system (GE Healthcare, Little Chalfont, UK). The column was then washed with 10 column volumes of 25 mM Tris–HCl pH 8.0, 150 mM NaCl before the target protein was eluted with the same buffer containing 10 mM L-glutathione. Fractions containing the brownish target protein were pooled. The protein was transferred into 50 mM HEPES, pH 7.5; 150 mM KCl using a HiTrap desalting column (GE Healthcare, Little Chalfont, UK). Protein concentrations were determined using the Bradford method [27 (link)]. The protein yield from cell cultures cultivated under anaerobic conditions was 0.006 µmol per l cell culture and from cell cultures cultivated under aerobic conditions 0.01 µmol per l cell culture.
8
Purification and Lyophilization of Trimerbody Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Harvested serum-free conditioned mammalian medium was centrifuged, 0.22 μm filtered (Nalgene, Neerijse, Belgium), concentrated (10x) with a 10.000 MWCO Vivaflow 50 filter (Vivascience GmbH, Hannover, Germany), dialyzed against PBS (pH7.4) and loaded onto a HisTrap HP 1 ml column using and ÄKTA Prime plus system (GE Healthcare, Uppsala, Sweden). The purified trimerbody was dialyzed against PBS, analyzed by SDS-PAGE under reducing conditions, and stored at −80°C. Harvested yeast medium was dialyzed against 50 mM Na3PO4 buffer, containing 100 mM NaCl (pH 8.0), 0.22 μm filtered and loaded onto a HisTrap HP 1 ml column using and ÄKTA Prime plus system. The purified trimerbody, was dialyzed against Na3PO4 buffer, analyzed by SDS-PAGE under reducing conditions, and stored at −80°C. For lyophilization, samples were dialyzed with 50 mM (NH4)HCO3 (pH 8.0), and lyophilized protein was stored at −20°C.
9
Purification and characterization of Bonsai
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ndc80bonsai (referred to as Bonsai) was a kind gift from A. Musacchio [11 (link)]. For anisotropy, Bonsai was purified as described below. Bonsai expression in E. coli BL21(DE3) was induced with 500 μM IPTG at OD600 = 0.45–0.7 for 16 h at RT. Cells were harvested by centrifugation at 3500 g. The bacterial pellet was resuspended in lysis buffer (50 mM Tris-HCl, pH 7.6, 300 mM NaCl, 1 mM DTT, 1 mM EDTA, Complete Protease Inhibitor Cocktail Tablets from Roche) and sonicated. After this, lysate was cleared by centrifugation at 50 000 × g for 60 min. Supernatant was filtered and Bonsai purified using ÄKTA prime plus system (GE Healthcare, Waukesha, WI, USA) and GST Sepharose Fast Flow (GE Healthcare). Cleavage buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM DTT, 1 mM EDTA) was used for equilibration and elution of Bonsai. PreScission protease (GE Healthcare) was applied into the column and left overnight at +4°C to cleave off GST followed by elution of Bonsai. Fractions containing Bonsai were collected and used for anisotropy. For GST-Aurora B production, BaculoGold™ expression system (BD Biosciences) was used to express Aurora B in Sf9 insect cells as described previously [60 (link)].
10
Size Exclusion Chromatography of Concentrated CM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FPLC was performed on an ÄKTA Prime plus system (GE Healthcare Life Sciences, London, UK). The concentrated CM was loaded onto a superose 12 10/300 GL column (GE Healthcare Life Sciences) equilibrated in phosphate-buffered saline. The column was eluted with two-column volumes of buffer. The fractions were filtered with a 0.22-μm syringe filter before using them for treatment of B cells.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!