Akta fplc

Manufactured by GE Healthcare

Sourced in United States, United Kingdom

The AKTA FPLC is a versatile laboratory instrument designed for fast protein liquid chromatography (FPLC) applications. It is capable of automating various chromatography techniques, such as ion exchange, size exclusion, and affinity chromatography, to facilitate the purification and analysis of biomolecules, including proteins, peptides, and nucleic acids.

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Lab products found in correlation

Superdex 200 10 300 gl column, by GE Healthcare (11 mentions) Optilab refractive index system, by Wyatt Technology (8 mentions) Hitrap, by Cytiva (5 mentions) Nanodrop, by Thermo Fisher Scientific (4 mentions) Complete mini, by Roche (4 mentions) Superdex 200 increase 10 300 gl column, by GE Healthcare (3 mentions) Superdex 200 10 300l column, by GE Healthcare (3 mentions) Superdex 200 size exclusion column, by GE Healthcare (3 mentions) Superdex 200, by GE Healthcare (3 mentions) Superdex 200 10 300 gl column, by Cytiva (3 mentions) Astra software package version 8, by Wyatt Technology (3 mentions) Superdex s200 resin, by GE Healthcare (3 mentions) Hiprep, by Cytiva (2 mentions) Superdex 75 column, by GE Healthcare (2 mentions) Edta free protease inhibitor mixture, by Roche (2 mentions) Blue dextran, by Merck Group (2 mentions) Optilab t rex, by Wyatt Technology (2 mentions) Blue dextran, by GE Healthcare (2 mentions) Aldolase, by GE Healthcare (2 mentions) Bovine rnase a, by GE Healthcare (2 mentions)

Spelling variants of this product

AKTA FPLC system (199 citations) AKTA Pure FPLC (35 citations) AKTA Purifier FPLC system (23 citations) AKTA UPC 10 FPLC system (4 citations) AKTA FPLC purification system (3 citations) AKTA FPLC machine (3 citations) AKTA Pure 25 FPLC system (3 citations) Akta Micro FPLC system (3 citations) AKTA Basic FPLC (3 citations) AKTA-FPLC 900 system (2 citations)

124 protocols using akta fplc

Recombinant Histone and CENP-A Nucleosome Assembly

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Drosophila or human core histones H2A, H2B, H3, H4 were produced as recombinant protein in E. coli BL21-CodonPlus(DE3)-RPIL (Novagen). Expressed histones were purified from the inclusion body using Hitrap SP column with AKTA FPLC (GE Healthcare) under denaturing condition^{36 (link)}. Human CENP-A was expressed with an N-terminal his6 tag in Alpha-Select Chemically Competent cell (Bioline). Expressed CENP-A was purified by Ni affinity chromatography using Ni-NTA beads (QIAGEN) under denaturing condtion^{18 (link)}. His6 tag were removed by thrombin protease (GE Healthcare) cleavage under non-denaturing condition (5 mM Tris–HCl, pH 7.4 and 5 mM 2-mercaptoethanol). All histones were further purified with a RP-protein column (Waters) and lyophilized. Nucleosomes were assembled by mixing histone octamer with DNA at 1:1.2 molar ratio in 10 mM Tris–HCl, pH 8.0, 1 mM EDTA (TE10/1) and 2 M NaCl, followed by dialysis in the same buffer with a gradual decrease in the NaCl concentration from 2 M to 0.25 M over 18 h. The CENP-A nucleosome assembled with human α-satellite DNA was heat re-positioned by incubating the reconstituted nucleosome at 37 °C for 3 h. All nucleosomes were purified from extra free DNA using DEAE-5PW column with AKTA FPLC (GE Healthcare)^{36 (link)}.

Zhou B.R., Yadav K.N., Borgnia M., Hong J., Cao B., Olins A.L., Olins D.E., Bai Y, & Zhang P. (2019). Atomic resolution cryo-EM structure of a native-like CENP-A nucleosome aided by an antibody fragment. Nature Communications, 10, 2301.

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Recombinant ALOD4 and OlyA Purification

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ALOD4 construct was generously provided by the lab of Dr Arun Radhakrishnan. Recombinant His-tagged ALOD4 and OlyA were purified as previously described (16 (link)). Briefly, ALOD4 expression was induced with 1 mM IPTG in OD_0.5 BL21 (DE3) pLysS E. coli for 16 h at 18°C. Cells were lysed and His-ALOD4 and His-OlyA were isolated by nickel purification followed by size exclusion chromatography (HisTrap-HP Ni column, Tricorn 10/300 Superdex 200 gel filtration column; FPLC AKTA, GE Healthcare). Protein-rich fractions were pooled and concentration was measured using a NanoDrop instrument.

Fowler J.W., Boutagy N.E., Zhang R., Horikami D., Whalen M.B., Romanoski C.E, & Sessa W.C. (2023). SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6. Journal of Lipid Research, 64(8), 100411.

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Purification of recombinant ALOD4 and OlyA

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ALOD4 and OlyA expression constructs were generously provided by the lab of Dr. Arun Radhakrishnan. Recombinant His-tagged ALOD4 and OlyA were purified as previously described (Endapally et al., 2019b (link)). Briefly, ALOD4 expression was induced with 1 mM IPTG in OD_0.5 BL21 (DE3) pLysS E. coli for 16 hr at 18 °C. Cells were lysed and His-ALOD4 and His-OlyA were isolated by nickel purification followed by size exclusion chromatography (HisTrap-HP Ni column, Tricorn 10/300 Superdex 200 gel filtration column; FPLC AKTA, GE Healthcare). Protein-rich fractions were pooled and concentration was measured using a NanoDrop instrument.

Fowler J.W., Zhang R., Tao B., Boutagy N.E, & Sessa W.C. (2022). Inflammatory stress signaling via NF-kB alters accessible cholesterol to upregulate SREBP2 transcriptional activity in endothelial cells. eLife, 11, e79529.

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Purification of GST-tagged Proteins

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Recombinant proteins were expressed in Rosetta™ (DE3) competent cells (EMD Millipore, Burlington, MA) in Miller LB supplemented with 100 μg/mL Ampicillin and 34 μg/mL Chloramphenicol at 37 °C until an OD₆₀₀ = 0.6–1.0 was reached; followed by induction with IPTG (1 mM) at 18 °C for 18 h. Cells were harvested by centrifugation and lysed (20 mM Tris-HCl pH 8.0, 200 mM NaCl, 5 mM DTT, Protease inhibitors). GST-tagged protein was immobilized on Pierce® glutathione agarose beads (Thermo Scientific, Waltham, MA) and washed (20 mM Tris-HCl pH 8.0 and 200 mM NaCl). Bound protein was eluted from the beads in wash buffer supplemented with 10 mM glutathione and concentration was determined using Bradford assay. Immobilized protein for crystallography was incubated with TEV at 4 °C for 18 h and subjected to size exclusion chromatography using a Superdex 75 Increase 10/300 GL (GE Healthcare, Chicago, IL) on an AKTA FPLC (GE Healthcare, Chicago, IL) in lysis buffer. The peak 215 nm fractions were collected. SDS-PAGE analysis was employed to determine purity, and protein was flash frozen and stored at −80 °C until needed.

Janezic E.M., Harris D.A., Dinh D., Lee K.S., Stewart A., Hinds T.R., Hsu P.L., Zheng N, & Hague C. (2019). Scribble co-operatively binds multiple α1D-adrenergic receptor C-terminal PDZ ligands. Scientific Reports, 9, 14073.

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Quantifying Nucleotide Extracts via AKTA FPLC

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An AKTA FPLC (GE Healthcare Life Sciences #18-1900-26) was used to quantify methanol-soluble extracts (Fig. 5). Five hundred-microliter samples were injected onto a Partisil 5 SAX anion-exchange column (4.6 mm × 250 mm; Whatman #4222-227). The nucleotides were separated using a gradient of 50% Buffer A (5 mM (NH₄)H₂PO₄ pH 3.8) and 50% Buffer B (0.25 M (NH₄)H₂PO₄, 0.5 M KCl pH 4.5) to 100% Buffer B for 30 min followed by an isocratic elution with 100% Buffer B for 15 min at a flow rate of 1.5 ml/min. The column was allowed to re-equilibrate to initial conditions for 5 min prior to the next injection. The absorbance of the nucleotides was determined at 280 nm.

Franklin D.A., He Y., Leslie P.L., Tikunov A.P., Fenger N., Macdonald J.M, & Zhang Y. (2016). p53 coordinates DNA repair with nucleotide synthesis by suppressing PFKFB3 expression and promoting the pentose phosphate pathway. Scientific Reports, 6, 38067.

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UBE3A HECT Domain Purification and Binding

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The HECT domain from WT UBE3A was cloned into pRSFDuet-1 (Novagen) with an Octa-histidine-GST double affinity tag fused to the N-terminus. A TEV protease cleavage sequence was introduced between His-GST and the HECT domain to excise the affinity tag. The UBE3A Q588E mutant was made by site-directed mutagenesis. Both wild type and UBE3A Q588E were transformed into E. coli BL21 codon plus RIL (Agilent) cells, and expression was induced by adding 0.6 mM IPTG at 16 °C for 18 h. UBE3A was purified on a nickel affinity gravity column followed by TEV protease digestion and size exclusion purification on an AKTA FPLC (GE healthcare). The purity of the fractions was confirmed by SDS-gel electrophoresis, combined, and concentrated to ~10 mg/mL for biophysical assays. N-terminal fluorescein-labeled ubiquitin (Anaspec) was prepared as described previously^{36 (link)}. Mixtures of 1 µM fluorescein-labeled ubiquitin and varied concentrations of UBE3A were prepared in a 384-well plate. Milli-fluorescence polarization (mP) values were detected by a Tecan Infinite M1000Pro plate reader at 30 °C. Iterative nonlinear curve fitting using a one-site specific binding model was applied in ProFit to obtain binding affinities (K_D).

Weston K.P., Gao X., Zhao J., Kim K.S., Maloney S.E., Gotoff J., Parikh S., Leu Y.C., Wu K.P., Shinawi M., Steimel J.P., Harrison J.S, & Yi J.J. (2021). Identification of disease-linked hyperactivating mutations in UBE3A through large-scale functional variant analysis. Nature Communications, 12, 6809.

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Prot B Purification and Fractionation

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Purified recombinant Prot B was fractionated in different chromatographic buffers (HEG + 0.02% NP-40, 0.2 mM PMSF, 0.5 mM benzamidine, 0 or 10 mM DTT, 150 or 500 mM NaCl) on an ∼24-mL Superdex 200 Increase 10/300 GL column on AKTA FPLC (GE Life Sciences). Typically, 0.5-mL starting material samples containing ∼0.5 mg of protein in a buffer with the NaCl concentration equal to that of the chromatographic buffer were injected on the column and fractionated isocratically at 0.4 mL/min, and 0.5-mL fractions corresponding to 0.3–1.0 column volumes were collected and analyzed by SDS-PAGE (in the absence of βME) and Coomassie staining. Molecular weights of fractionating proteins were assigned based on the column calibration by the manufacturer (GE Life Sciences).

Emelyanov A.V, & Fyodorov D.V. (2016). Thioredoxin-dependent disulfide bond reduction is required for protamine eviction from sperm chromatin. Genes & Development, 30(24), 2651-2656.

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Purification of EGFP-Tf Receptor Chimera

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HEK-293S GnTI^- cells were transiently transfected with EGFP-receptor fusion plasmid through transient transfection with PEI MAX 40K reagent (Polysciences, Inc.) according to manufacturer’s procedure and were harvested 48 hours post-transfection. (We note that the GnTI^- subtype does not produce complex glycosylation modifications and previous studies have shown Tf-binding to the glycoproteins, TfR1 and TfR2, is not glycosylation dependent ^{18 (link)–20 (link)}). Cells were lysed and membrane proteins solubilized by addition of 20 mM HEPES, pH 7.4, 150 mM NaCl, 1% laurylmaltose neopentyl glycol (LMNG), and protease inhibitor cocktail (Biotools). Solubilization of the EGFP-TfR2-TfR1HD chimera required the addition of 10 μΜ holo- Tf. Lysates were rotated for one hour at 4 °C. Insoluble material was removed by ultracentrifugation at 70,000 xG for 40 minutes at 4 °C. Solubilized protein was applied to a AKTAFPLC (G.E. Healthcare) with in-line Superose 6 Increase gel filtration column and Waters 474 scanning fluorescence detector (excitation: 488 nm, emission: 520 nm). The running buffer was 20 mM HEPES, pH 7.4, 150 mM NaCl, 0.01% LMNG. The fraction containing maximal GFP fluorescence was collected for use in SPR binding studies.

Kleven M.D., Jue S, & Enns C.A. (2018). The Transferrin Receptors, TfR1 and TfR2 Bind Transferrin through Differing Mechanisms. Biochemistry, 57(9), 1552-1559.

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Plasma Protein Separation by FPLC

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Plasma proteins of pooled plasma samples were separated using S6-superose sizing columns on AKTA FPLC (GE Healthcare).

Wagner T., Bartelt A., Schlein C, & Heeren J. (2015). Genetic Dissection of Tissue-Specific Apolipoprotein E Function for Hypercholesterolemia and Diet-Induced Obesity. PLoS ONE, 10(12), e0145102.

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Preparation and Characterization of BODIPY-Labeled α-Synuclein

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BODIPY-labelled α-Syn was prepared as described previously [26 (link), 27 (link)]. Briefly, E114C mutant α-Syn was expressed in BL21 (DE3) E.coli and purified using Ni–NTA resin (Gold Biotech), dialyzed, and alkylated with BODIPY-Fl maleimide (Lumiprobe). The labelled protein was purified over a HiTrap Q HP column on an AKTA FPLC (GE Life Sciences) and characterized by MALDI-TOF mass spectrometry. To generate our 25% fluorescently labelled aggregates, 70 ng/µL WT monomer plus 25 ng/µL of BODIPY-E114C monomer were incubated with either 5 ng/µL PFF or 5 ng/µL of patient derived LB α-Syn and amplified as described above.

Marotta N.P., Ara J., Uemura N., Lougee M.G., Meymand E.S., Zhang B., Petersson E.J., Trojanowski J.Q, & Lee V.M. (2021). Alpha-synuclein from patient Lewy bodies exhibits distinct pathological activity that can be propagated in vitro. Acta Neuropathologica Communications, 9, 188.

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