Q sepharose fast flow

Manufactured by GE Healthcare

Sourced in United States, United Kingdom, Sweden

Q Sepharose Fast Flow is a chromatography resin used for the purification and separation of biomolecules. It is composed of highly cross-linked agarose beads with quaternary ammonium ligands, which provide strong anion exchange functionality. The resin is designed for high-performance protein and nucleic acid purification in both batch and column formats.

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Lab products found in correlation

Superdex 200, by GE Healthcare (7 mentions) Pullulan standards, by Resonac (4 mentions) Ni nta affinity column, by Qiagen (3 mentions) Heparin, by Merck Group (3 mentions) At 3, by Werfen (3 mentions) Hc 2, by Hyphen Biomed (3 mentions) Sephacryl s 400 hr, by GE Healthcare (3 mentions) D glucuronic acid, by Merck Group (3 mentions) L rhamnose, by Merck Group (3 mentions) D xylose, by Merck Group (3 mentions) Sephacryl s 100 hr, by GE Healthcare (2 mentions) Deae sepharose fast flow, by GE Healthcare (2 mentions) Kta purifier fplc system, by GE Healthcare (2 mentions) Ni sepharose 6 fast flow, by GE Healthcare (2 mentions) Ni affinity, by Merck Group (2 mentions) Econo pac column, by Bio-Rad (2 mentions) Histrap hp, by GE Healthcare (2 mentions) D galactose, by Merck Group (2 mentions) D galacturonic acid, by Merck Group (2 mentions) L arabinose, by Merck Group (2 mentions)

62 protocols using q sepharose fast flow

Engineered Pro-TGF-β1 and αVβ6 Headpiece Production

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The human pro-TGF-β1 construct contains an N-terminal 8-His tag, followed by a SBP tag and a 3C protease site. A C4S mutation, an R249A furin cleavage site mutation and N-glycosylation site mutations N107Q and N147Q were introduced to facilitate protein expression, secretion and crystallization. Pro-TGF-β1 was expressed in CHO Lec 3.2.8.1 cells utilizing the pEF-puro vector, purified in three steps as described in ^{1 (link)} and yielded 1 mg purified protein per liter culture supernatant. The same protein was used in EM, SAXS, HDX, and surface plasmon resonance.
Soluble α_Vβ₆ headpiece was prepared as in ^{2 (link)}. The α_Vβ₆ head uses the same α_V construct as in the α_Vβ₆ headpiece and the β₆ βI domain (residues 108–352) with I270C mutation followed by a 6X His tag. Proteins expressed in HEK293S Gnt I⁻ cells with Ex-Cell 293 serum free media (Sigma) were purified using Ni-NTA affinity column (Qiagen). Protein was cleaved with 3C protease at 4 °C overnight and passed through Ni-NTA resin and further purified using an ion exchange gradient from 50 mM NaCl to 1M NaCl, 20 mM Tris-HCl, pH 8.0 (Q fast-flow Sepharose, GE healthcare) and gel filtration (Superdex 200, GE healthcare). Cell lines were originally from American Type Culture Collection (ATCC) and not authenticated or tested for mycoplasma contamination.

Dong X., Zhao B., Iacob R.E., Zhu J., Koksal A.C., Lu C., Engen J.R, & Springer T.A. (2017). Force Interacts with Macromolecular Structure in Activation of TGF-β. Nature, 542(7639), 55-59.

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Purification of αvβ6 Integrin Headpiece

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Soluble αvβ6 headpiece was prepared as described earlier15 (link). Briefly, the αv headpiece construct contained residues 1–594 of αv with an M400C mutation followed by a 3C protease site, the ACID coiled coil, a strep II tag and a 6 × histidine tag. β6 headpiece residues 1–474 with an I270C mutation or β3 headpiece residues 1–472 with a Q267C mutation, were followed by a 3C site, BASE coiled coil, and a histidine tag. The mutations generated a disulfide bond creating an α/β heterodimer. Proteins were co-expressed in HEK293S Gnt I− cells31 (link) with Ex-Cell 293 serum-free medium (Sigma) and purified with Ni-NTA affinity columns (Qiagen). Protein was cleaved by 3C protease at 4 °C overnight, passed through Ni-NTA resin and further purified with an ion-exchange gradient from 50 mM to 1 M NaCl, 20 mM Tris-HCl, pH 8.0 (Q fast-flow Sepharose, GE Healthcare) and finally gel filtrated (Superdex 200, GE Healthcare).

Kotecha A., Wang Q., Dong X., Ilca S.L., Ondiviela M., Zihe R., Seago J., Charleston B., Fry E.E., Abrescia N.G., Springer T.A., Huiskonen J.T, & Stuart D.I. (2017). Rules of engagement between αvβ6 integrin and foot-and-mouth disease virus. Nature Communications, 8, 15408.

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Purification of Therapeutic Proteins

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Culture media were centrifuged at 500g for 20 min and filtered (0.45 μm). Purification of IgG1 and
GLA, GBA, and EPO was performed as reported in previous works.^{27 (link),37 (link),38 (link)} For AGA, 20% (v/v) conditioning
buffer (70 mM Tris–HCl, pH 7.0) was added to the media and
loaded on a column packed with Q-FastFlow Sepharose (GE Healthcare)
pre-equilibrated with 5 column volumes (CV) of equilibration buffer
(20 mM Tris–HCl, 20 mM sodium acetate, 70 mM sodium chloride,
pH 6.8). After washing the column with 6 CV of wash buffer (20 mM
Tris–HCl, 20 mM sodium acetate, 70 mM sodium chloride, pH 6.8),
the enzyme was one-step eluted with elution buffer (25 mM sodium acetate,
250 mM NaCl, pH 4.5) into a tube containing 300 mM sodium phosphate
(pH 7.3). The eluates were diluted with 50% (v/v) 4 M (NH₄)₂SO₄ and further loaded on a Phenyl-Sepharose
Fast Flow (high substitution) column (GE Healthcare). After washing
and equilibrating the column with 5 CV of 2 M (NH₄)₂SO₄ and 20 mM Tris–HCl (pH 7.0), the enzyme
was eluted with elution buffer in a gradient (from 2 M to 0 M (NH₄)₂SO₄, 20 mM Tris–HCl, pH 7.0).

García-García A., Serna S., Yang Z., Delso I., Taleb V., Hicks T., Artschwager R., Vakhrushev S.Y., Clausen H., Angulo J., Corzana F., Reichardt N.C, & Hurtado-Guerrero R. (2021). FUT8-Directed Core Fucosylation of N-glycans Is Regulated by the Glycan Structure and Protein Environment. ACS Catalysis, 11(15), 9052-9065.

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Soluble αVβ6 Integrin Headpiece Purification

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Soluble α_Vβ₆ headpiece was prepared similarly as in ref. 33 (link). In brief, the α_V headpiece (residues 1–594) with the M400C mutation was followed by a 3C protease site, the ACID coiled coil, a strep II tag and a histidine tag. β₆ headpiece residues 1–474 with I270C or β₃ headpiece residues 1–472 with Q267C were followed by the 3C site, the BASE coiled coil, and a histidine tag. The cysteine mutations generated a disulfide bond that prevented α/β subunit dissociation. Proteins expressed in HEK293S GnTI⁻ cells with Ex-Cell 293 serum-free medium (Sigma) were purified with Ni-NTA affinity columns (Qiagen). Protein was cleaved by 3C protease at 4 °C overnight, passed through Ni-NTA resin and further purified with an ion-exchange gradient from 50 mM to 1 M NaCl, 20 mM Tris-HCl, pH 8.0 (Q fast-flow Sepharose, GE Healthcare) and gel filtration (Superdex 200, GE Healthcare).

Dong X., Hudson N.E., Lu C, & Springer T.A. (2014). Structural determinants of integrin β-subunit specificity for latent TGF-β. Nature structural & molecular biology, 21(12), 1091-1096.

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Constructing a Fusion Enzyme for Enhanced Cellulose Degradation

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Sequence alignment of MtLPMO9G and MtLPMO9L was performed by ClustalW software (http://www.genome.jp/tools-bin/clustalw) (55 (link)), and a more intuitive alignment was presented by ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) (56 (link)). Next, the non-CD module (AA206–AA284) of MtLPMO9G was fused to the C terminus of MtLPMO9L to construct a fusion form (MtLPMO9L-CBM) (refer to Supporting information Table S2 for primer details). The recombinant enzymes, MtLPMO9L and MtLPMO9L-CBM, were recombinantly overexpressed as secreted proteins in P. pastoris X-33 and purified by anion chromatography using Q-Sepharose fast-flow (GE Healthcare) (57 ). The Purified enzymes were saturated with CuSO₄ and desalted using an Amicon ultracentrifugation device equipped with a 10 kDa cut-off membrane, as previously described by R. Kont et al. (40 (link)). Furthermore, the three-dimensional structures of MtLPMO9L and MtLPMO9L-CBM were predicted based on their amino acid sequences using Robetta server (50 (link)) with the method of RoseTTAFold (51 (link)).

Gao W., Li T., Zhou H., Ju J, & Yin H. (2023). Carbohydrate-binding modules enhance H2O2 tolerance by promoting lytic polysaccharide monooxygenase active site H2O2 consumption. The Journal of Biological Chemistry, 300(1), 105573.

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Purification and Identification of Yeast tRNA-Binding Proteins

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A yeast cytosolic fraction was prepared from logarithmically growing the S. cerevisiae strain W303-1A. First, the yeast cells were converted into spheroplasts and disrupted in 50 mM Tris–HCl, pH 7.4, 350 mM NaCl, 5 mM MgCl₂ supplemented with 0.5 mM PMSF, a 1/1000 volume of PIC (Roche Diagnostics), and 1.0 mM β-mercaptoethanol by vigorous agitation with glass beads. The lysate was mixed with final 0.5% wt/vol of Triton X-100, and was centrifuged at 100,000×g for 30 min. The recovered supernatant was passed through Q-Sepharose Fast Flow (GE Healthcare) to remove endogenous tRNAs. The flow-through fraction was applied to the tRNA-resin prepared as described previously in the presence or absence of 3 mM Mg-ATP, and bound proteins were eluted with 50 mM Tris–HCl, pH 7.4, 1.5 M NaCl, 5 mM MgCl₂ after extensive washing. ATP-dependent or ATP-sensitive tRNA-binding proteins were identified by peptide mass fingerprinting with a Voyager DE MALDI/TOF mass spectrometer (Applied Biosystems, Foster City, CA).

Takano A., Kajita T., Mochizuki M., Endo T, & Yoshihisa T. (2015). Cytosolic Hsp70 and co-chaperones constitute a novel system for tRNA import into the nucleus. eLife, 4, e04659.

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Purification of Srt-VSG3 Protein from T. brucei

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Srt-VSG3 protein was purified from T. brucei PLC WT expressing Srt-VSG3 as described elsewhere.^{14 (link)} Briefly, cells were cultured in vitro in HMI-9 media to a density of 4 × 10⁶ cells/mL. Cells were pelleted, lysed in 0.2 mM ZnCl₂ + HALT protease inhibitor and then the lysis mixture was centrifuged at 10,000xg for 10 min. The pellet which contained the membrane material was resuspended in pre-warmed (40°C) 20 mM HEPES pH 7.5 with 150 mM sodium chloride (NaCl), enabling the activation of endogenous lipases and resulting in the efficient release of surface VSG protein from the membrane. The membranous material was then pelleted two more times, while supernatants (containing soluble VSG) were collected. Supernatants were loaded onto an anion-exchange column (Q-Sepharose Fast-Flow, GE Healthcare), which had been equilibrated with 20 mM HEPES buffer with 150 mM NaCl (the VSG does not bind to the column, while contaminating proteins are trapped). Srt-VSG3 was then separated from remaining contaminants and aggregation products via a gel filtration column (Superdex 200, GE Healthcare) equilibrated in 20 mM HEPES buffer with 150 mM NaCl. Aliquots from the gel filtration runs were analyzed on SDS–PAGE for visual inspection (Figure S2B).

Rajantie I., Ekman N., Iljin K., Arighi E., Gunji Y., Kaukonen J., Palotie A., Dewerchin M., Carmeliet P, & Alitalo K. (2001). Bmx Tyrosine Kinase Has a Redundant Function Downstream of Angiopoietin and Vascular Endothelial Growth Factor Receptors in Arterial Endothelium. Molecular and Cellular Biology, 21(14), 4647-4655.

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NanoLuc Expression and Purification

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E. coli BL21-CodonPlus (DE3) (Novagen, Sigma-Aldrich, St. Louis, MO, USA) transformed with plasmid pET22b-NLuc carrying the NanoLuc gene, codon-optimized for bacterial expression (Evrogen, Moscow, Russia) were cultivated at 37 °C in LB medium, containing 200 µg mL⁻¹ ampicillin and induced by 1 mM IPTG after reaching an OD₅₉₀ of 0.6–0.7, then further cultivated for 3 h. The cells harvested by centrifugation were resuspended in 1 mM TrisHCl, pH 8.0, destroyed by ultrasonication on ice, and then centrifuged. Streptomycin sulfate (1%) was added to the supernatant, and the formed precipitate was separated by centrifugation. Supernatant was dialyzed against 20 mM TrisHCl, pH 8.0, loaded onto the column Q Sepharose Fast Flow (GE Healthcare, Chicago, IL, USA), and proteins were eluted with NaCl gradient (0–0.3 M).

Kudryavtsev A.N., Krasitskaya V.V., Efremov M.K., Zangeeva S.V., Rogova A.V., Tomilin F.N, & Frank L.A. (2023). Ca2+-Triggered Coelenterazine-Binding Protein Renilla: Expected and Unexpected Features. International Journal of Molecular Sciences, 24(3), 2144.

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Purification of Recombinant Proteins

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TAP-ANV and ANV-6L15 were cloned into the expression vector pET20b(+)
(Novagen, Madison, WI, USA) and expressed in Escherichia coliBL21(DE3)pLysS as described before [34 (link)].
The recombinant proteins were purified by chromatography on Q-Sepharose™
fast flow (GE Healthcare, Piscataway, NJ).

Yeh Y.H., Chang S.H., Chen S.Y., Wen C.J., Wei F.C., Tang R., Achilefu S., Wun T.C, & Chen W.J. (2017). Bolus injections of novel thrombogenic site-targeted fusion proteins comprising annexin-V and Kunitz protease inhibitors attenuate intimal hyperplasia after balloon angioplasty. International journal of cardiology, 240, 339-346.

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Purification of Mutant Cytochrome c Peroxidase

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Mutations were made to an E. coli codon-optimized gene for cytochrome c peroxidase by site-directed mutagenesis via PCR with primers containing the desired mutant sequence. The gene was contained within a pET-17b plasmid bearing an ampicillin resistance cassette. Mutant proteins were transformed into BL21-DE3 high-efficiency chemically competent E. coli (New England Biolabs) and expressed and purified by a protocol previously described and modified by our lab.^{28 (link),72 (link),75 (link)} Briefly, cells were cultured overnight in LB (rich) media, induced with 0.5 mM IPTG, and pelleted after 3–4 h of expression. Cell pellets were lysed by sonic disruption, and the clarified soluble supernatant was subjected to anion exchange chromatography (DEAE Sepharose Fast Flow anion exchange resin, GE Healthcare) in phosphate buffer (50 mM, 1 mM EDTA, pH 7.0) eluted on a linear gradient of potassium chloride (0–500 mM) followed by gel filtration chromatography (Sephacryl S-100 HR, GE Healthcare) in potassium phosphate buffer (100 mM, pH 7.0). The major peak fractions were pooled, assessed for purity by mass spectrometry (ESI-MS), and further purified as necessary by separation on Q Sepharose Fast Flow (GE Healthcare) strong anion exchange resin (eluted on a linear potassium chloride gradient) until the hemoprotein content was minimal.

Mirts E.N., Dikanov S.A., Jose A., Solomon E.I, & Lu Y. (2020). A Binuclear CuA Center Designed in an All α-Helical Protein Scaffold. Journal of the American Chemical Society, 142(32), 13779-13794.

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