Kta protein purification system

Manufactured by GE Healthcare

Sourced in United States

The ÄKTA protein purification system is a versatile and automated chromatography system designed to purify a wide range of biomolecules, including proteins, peptides, and nucleic acids. The system offers precise control over the purification process, enabling researchers to achieve high-purity samples with consistent results.

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19 protocols using kta protein purification system

SCX Fractionation of Peptides

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Strong cation exchange (SCX) fractionation was performed as in (Gruhler et al., 2005 (link)) with some modifications. Doubly digested mixed lysate was fractionated using a Resource S SCX column (1 mL, GE Healthcare) in a ÄKTA protein purification system (GE Healthcare). Briefly, the mixed lysate was loaded onto the SCX column equilibrated in Solvent A (5 mM potassium dihydrogen phosphate, 30% acetonitrile (ACN; Fisher Chemicals, A955-212), pH 2.7 with TFA) at a flow rate of 1 mL/min. Flow-through during loading was kept for subsequent phosphopeptide enrichment. Peptides bound to the column were eluted as 2 mL fractions with a 0%–50% linear gradient of Solvent B (350 mM potassium chloride, 5 mM potassium dihydrogen phosphate, 30% ACN, pH 2.7 using TFA) at a flow rate of 1 mL/ min, over a period of 30 min. Dilute fractions, based on estimation from the chromatogram, were pooled together for subsequent phosphopeptide enrichment.

Tay Y.D., Leda M., Spanos C., Rappsilber J., Goryachev A.B, & Sawin K.E. (2019). Fission Yeast NDR/LATS Kinase Orb6 Regulates Exocytosis via Phosphorylation of the Exocyst Complex. Cell Reports, 26(6), 1654-1667.e7.

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Purification of DcpS Enzyme from E. coli

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Human or C. elegans DcpS was expressed
from the respective
pET vectors in the Rosetta 2(DE3) Escherichia coli strain. Protein expression was induced with 0.4 mM IPTG when OD₆₀₀ reached 0.5–0.8, and then, cells were further incubated
at 18 °C overnight. After expression, the bacterial pellet was
collected by centrifugation (7000g, 10 min) and was
frozen at −80 °C. The frozen pellet was thawed on ice
and then resuspended in ice-cold lysis buffer: 50 mM phosphate buffer
pH 7.2, 150 mM NaCl, 1% Triton X-100, 20 mM imidazole. Suspension
was sonicated and then centrifuged for 2 h. The supernatant was loaded
on the HisTrapHP column (GE Healthcare Life Sciences). All separation
by affinity chromatography was done with a gradient of imidazole 20–600
mM in 50 mM phosphate buffer pH 7.2. The DcpS protein was further
purified and buffer exchanged: 50 mM Tris-HCl, 150 mM NaCl, 10% glycerol
pH 7.5, by gel filtration (“Superdex 200 10/300GL” GE
Healthcare Life Sciences) using an ÄKTA protein purification
system (GE Healthcare Life Sciences) and stored in −80 °C. A. suum DcpS was prepared according to the procedure
described previously.^{23 (link)}

Pietrow P., Ferenc-Mrozek A., Piecyk K., Bojarska E., Darzynkiewicz E, & Jankowska-Anyszka M. (2019). Decapping Scavenger Enzyme Activity toward N2-Substituted 5′ End mRNA Cap Analogues. ACS Omega, 4(17), 17576-17580.

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Characterization of FP Complex Formation

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All SEC experiments were performed on a GE ÄKTA protein purification system using a Superdex 200 column at a flow rate of 0.2 mL/min. Affinity-purified Dronpa and TagRFP-T protein stock solutions were first further purified individually using SEC. The pure fractions of each FP were then pooled, mixed at a 1:1 molar ratio, and concentrated to a total protein concentration of approximately 120 μM; the stoichiometry was verified at 1.00:1 using absorption spectroscopy followed by spectral deconvolution. A total of 150 μL of the mixed stock solution was subsequently examined with the same SEC parameters to resolve any additional oligomeric states present in the concentrated mixture (Supplementary Fig. 2A). Collected fractions were subjected to absorption spectroscopy and spectral deconvolution in order to determine the concentration of Dronpa and TagRFP-T in each. A peak at approx. 92 kDa that was not observed in the chromatographs of the individual FPs was found to contain a stoichiometric ratio of approximately 2.5 TagRFP-T molecules to 1 Dronpa. It is known that TagRFP-T forms dimers at high concentrations. Since Dronpa and TagRFP-T are each 30kDa, both size and stoichiometry verified the presence of a trimeric FP complex between a TagRFP-T dimer and a Dronpa monomer.

Mo G.C., Ross B., Hertel F., Manna P., Yang X., Greenwald E., Booth C., Plummer A.M., Tenner B., Chen Z., Wang Y., Kennedy E.J., Cole P.A., Fleming K.G., Palmer A., Jimenez R., Xiao J., Dedecker P, & Zhang J. (2017). Genetically-Encoded Biosensors for Visualizing Live-cell Biochemical Activity at Superresolution. Nature methods, 14(4), 427-434.

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Anti-SEMA3A Antibody Production

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The Expi293F expression system (Thermo Fisher Scientific) was used for anti-SEMA3A IgG production. Using HiTrap protein G HP (5 mL) of ÄKTA Protein Purification System (GE Healthcare, Barrington, IL) purified anti-SEMA3A antibodies were concentrated by Amicon Ultra Centrifugal Filter (Merck Millipore, Burlington, MA). The produced anti-SEMA3A IgGs were evaluated by size exclusion high-performance liquid chromatography analysis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for aggregation and degradation. in vivo experiment was further tested by LAL endotoxin quantitation kit (QCL-1000, Lonza, Basel, Switzerland).

Lee J., Shin Y.J., Lee K., Cho H.J., Sa J.K., Lee S.Y., Kim S.H., Lee J., Yoon Y, & Nam D.H. (2017). Anti-SEMA3A Antibody: A Novel Therapeutic Agent to Suppress Glioblastoma Tumor Growth. Cancer Research and Treatment : Official Journal of Korean Cancer Association, 50(3), 1009-1022.

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Purification and Characterization of Anti-FGFR3 scFv Antibodies

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The antibody variable region of isolated FGFR3-specific scFv was analyzed using phagemid vectors. The variable region sequences of heavy chain (IgG1) or light chain isolated from phagemid vectors were inserted into each mammalian expression vectors, respectively. Anti-FGFR3 antibodies were produced using the Expi293 transient mammalian expression system (Gibco, A14635, Carlsbad, CA, USA) through co-transfection of the above-mentioned vectors. Following transfection, the culture supernatant was purified using the ÄKTA protein purification system (GE Healthcare Life Sciences, Uppsala, Sweden) with HiTrap Mabselect SuRe (GE Healthcare Life Sciences, 11-0034-93, Uppsala, Sweden). After purification, enrichment was performed with Amicon^® Ultra Centrifugal Filter (Merck Millipore, MA, USA). The characteristics of the highly purified antibodies were analyzed using SDS-PAGE and SEC-HPLC.

Min B., Yoo M., Kim H., Cho M., Nam D.H, & Yoon Y. (2021). Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody. International Journal of Molecular Sciences, 22(12), 6240.

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Recombinant PDF Protein Expression and Purification

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The pdf gene was amplified from V. anguillarum YN genome DNA by PCR using the following primers: For: 5′-CGCGGATCCATGTCTGTATTACAAG-3′ (the underlined region indicates BamH I site) and Rev: 5′-CCGCTCGAGTTAGTTTTTTTCGTTATAG-3′ (the underlined region indicates Xho I site). PCR products were cloned into pMD18-T vector (TaKaRa). After sequence confirmation, PCR products were inserted in the multiple cloning site of vector pET30a(+) (Novagen) and the resulting plasmid was designated as pET30a(+)::pdf. Plasmid pET30a(+)::pdf was transformed into E. coli BL21(DE3) cells. Recombinant PDF was expressed and purified as follows. Briefly, cells harboring plasmids pET30a(+)::pdf were grown to an absorbance at 600 nm (A₆₀₀) of 0.6 and induced with 0.5 mM isopropyl-D-thiogalactopyranoside at 16°C overnight. Cells were harvested by centrifugation, washed in HEPES buffer (25 mM, pH 7.4) and resuspended in HEPES (pH 7.4)-75 mM KCl-10% glycerol (buffer A). Then cells were lysed by sonication and centrifugated at 25,000 ×g. The supernatant was loaded onto a 5 ml HisTrap FF column (GE healthcare) and equilibrated in buffer A. The column was further washed and eluted with a gradient of imidazole from 0 to 300 mM using ÄKTA protein purification system (GE healthcare).

Yang N, & Sun C. (2016). The Inhibition and Resistance Mechanisms of Actinonin, Isolated from Marine Streptomyces sp. NHF165, against Vibrio anguillarum. Frontiers in Microbiology, 7, 1467.

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Overexpression and Purification of DHFR Mutants

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WT DHFR and all mutants used in this study were cloned into a pET24 expression vector and overexpressed in the BL21(DE3) pLys E. coli strain.
A single colony of the transformed E. coli carrying the wild type or mutation dhfr was cultured in Luria-Bertani liquid medium containing 50 μg/mL kanamycin (LB-kana) at 30°C overnight, and then inoculated to fresh LB-kana (1:100 dilution) and incubated again at 30°C. When the OD₆₀₀ of the culture reached 0.6, isopropyl β-D-1-thiogalactopyranoside (final concentration, 0.4 mM) was added. Cultures were incubated for an additional 12–16 h at 25°C. The cells were then collected by centrifugation and disrupted by sonication. The recombinant proteins were purified with Ni-NTA Superflow (QIAGEN, U.S.) according to the manufacturer’s instructions. Then, the collected protein sample was run with Superdex 75pg Column and was desalted with the desalting Column in ÄKTA protein purification system (GE Healthcare, U.S.). The final concentration of the purified protein was determined using the BCA protein assay kit (PIERCE CHEMICAL, USA) or the NanoDrop instrument (GE Healthcare, U.S.).

Tian J., Woodard J.C., Whitney A, & Shakhnovich E.I. (2015). Thermal Stabilization of Dihydrofolate Reductase Using Monte Carlo Unfolding Simulations and Its Functional Consequences. PLOS Computational Biology, 11(4), e1004207.

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Yeast Protein Purification via Affinity Chromatography

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The purification of yeast supernatants (1 liter) obtained from scaled up cultures, was performed by Affinity Chromatography, using an ÄKTA Protein Purification System (Ge Healthcare Life Sciences, Illinois, USA) using a HisTrap HP 1 ml column (Ge Healthcare Life Sciences, Illinois, USA). Wash steps and equilibration were performed according to the manufacturer’s instructions, using Wash buffer (20 mM sodium phosphate, 500 mM NaCl, pH 7.3). Elution was performed through a linear gradient of Elution buffer (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.3). Chromatogram analysis was accomplished using UNICORN 7.0 software.

Duranti C., Carraresi L., Sette A., Stefanini M., Lottini T., Crescioli S., Crociani O., Iamele L., De Jonge H., Gherardi E, & Arcangeli A. (2018). Generation and characterization of novel recombinant anti-hERG1 scFv antibodies for cancer molecular imaging. Oncotarget, 9(79), 34972-34989.

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Protein Purification via Chromatography

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Fast performance liquid chromatography was performed on a GE Healthcare Äkta Protein Purification System. Nickel affinity chromatography was performed with a HisTrap Excel 5-ml column (GE Healthcare). Gel filtration was performed by hand with disposable Sephadex® G-25 columns (GE Healthcare).

Holder P.G., Jones L.C., Drake P.M., Barfield R.M., Bañas S., de Hart G.W., Baker J, & Rabuka D. (2015). Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion. The Journal of Biological Chemistry, 290(25), 15730-15745.

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Gateway Protein Expression and Purification

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Genes were transferred from pENTR into pDEST-17 using the Gateway recombinase system (Life Technologies). Proteins were expressed and purified as previously described (6 (link)). In short, E. coli Rosetta cells were induced at 30°C with a final concentration of 0.1 mM IPTG for 3 h. His-tagged proteins were purified by affinity chromatography with an ÄKTA Protein Purification System (GE Life Sciences) using a HisTrap TALON crude column (GE Life Science).

de Lange O., Wolf C., Thiel P., Krüger J., Kleusch C., Kohlbacher O, & Lahaye T. (2015). DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats. Nucleic Acids Research, 43(20), 10065-10080.

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