Sp sepharose ff

Manufactured by GE Healthcare

Sourced in United States, Sweden

SP Sepharose FF is a strong cation exchange media used for the purification of biomolecules. It consists of cross-linked agarose beads with sulfopropyl (SP) functional groups. This media is designed for high-performance chromatographic separations and can be used for the capture and purification of proteins, peptides, and other charged biomolecules.

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12 protocols using sp sepharose ff

Purification of Thermus thermophilus RNAP

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T. thermophilus RNAP core enzyme was prepared from T. thermophilus strain H8 (DSM 579; DSMZ), using cell lysis, polyethylenimine precipitation, ammonium sulfate precipitation, cation-exchange chromatography on SP Sepharose FF (GE Healthcare), anion-exchange chromatography on Mono Q 10/100 GL (GE Healthcare), and cation-exchange chromatography on Mono S HR (GE Healthcare), as in Zhang et al., 2012 (link) and Maffioli et al., 2017 (link).
T. thermophilus σ^A was prepared by co-expression of a gene for N-terminally hexahistidine-tagged T. thermophilus σ^A in E. coli, followed by cell lysis, immobilized-metal-ion affinity chromatography on Ni-NTA agarose (Qiagen) and anion-exchange chromatography on Mono Q (GE Healthcare), as in Zhang et al., 2012 (link).
T. thermophilus RNAP σ^A holoenzyme was prepared by combining T. thermophilus RNAP core enzyme and T. thermophilus σ^A, followed by size-exclusion chromatography on Superdex 200 (GE Healthcare), as in Zhang et al., 2014 (link).

Lin W., Das K., Degen D., Mazumder A., Duchi D., Wang D., Ebright Y.W., Ebright R.Y., Sineva E., Gigliotti M., Srivastava A., Mandal S., Jiang Y., Liu Y., Yin R., Zhang Z., Eng E.T., Thomas D., Donadio S., Zhang H., Zhang C., Kapanidis A.N, & Ebright R.H. (2018). STRUCTURAL BASIS OF TRANSCRIPTION INHIBITION BY FIDAXOMICIN (LIPIARMYCIN A3). Molecular cell, 70(1), 60-71.e15.

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Purification and Characterization of Momordica charantia L. Seed Proteins

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Momordica charantia L. seeds were obtained from the Institute of Agricultural Science and Technique of Sichuan Province, China. Electrophoresis reagents were products of Sigma-Aldrich (St Louis, MO). Macro-Cap-SP, SP-Sepharose FF, Sephacryl S-100HR and ampholyte were purchased from GE Healthcare Bio-Sciences AB (Uppsala, SE). Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) used in cell culture were from Gibco BRL (Grand Island, NE). The lung adenocarcinoma A549 cell line was obtained from American Type Culture Collection (ATCC CCL-185) (Manassas, VA). LMW Calibration Kit was supplied by SIBAS (Shanghai, China). pUC18 DNA used in detection of topological activity was obtained from TAKARA (Dalian, China). All other chemical reagents were standard commercial products of analytical grade.

Meng Y., Lin S., Liu S., Fan X., Li G, & Meng Y. (2014). A Novel Method for Simultaneous Production of Two Ribosome-Inactivating Proteins, α-MMC and MAP30, from Momordica charantia L. PLoS ONE, 9(7), e101998.

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Evaluation of Strong Cation Exchange Chromatography Resins

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Prepacked 25 mm × 7 mm i.d. HiTrap columns containing SP Sepharose FF, SP Sepharose XL and Capto S, all strong cation exchangers, were purchased from GE Healthcare Life Sciences. A prepacked 50 mm × 5 mm i.d. S HyperCel column was obtained from Pall Life Sciences. The physical properties of the resins are summarized in Table 2. SP FF comprises an agarose base matrix functionalized with sulfonate groups attached to a short spacer arm. SP XL uses the same agarose base matrix as SP FF, but also contains covalently attached 40 kDa dextran extenders. Both the dextran and the agarose base matrix carry sulfonate functionalization, substantially expanding the volume available for adsorption within this material as compared to its non-polymer-modified counterpart but concomitantly reducing the pore space available for transport. Capto S contains similar 40 kDa extenders to those present in SP XL but the cross-linking density of the agarose base matrix is higher than for the Sepharose materials [7 ], resulting in a slightly smaller pore lumen [16 (link)]. S HyperCel is comprised of a fully cellulosic base matrix without any defined polymer modification [8 (link)], but it exhibits properties comparable to those seen in polymer-modified stationary phases [17 ], including increased capacity and enhanced uptake rates.

Angelo J.M, & Lenhoff A.M. (2016). Determinants of Protein Elution Rates from Preparative Ion-Exchange Adsorbents. Journal of chromatography. A, 1440, 94-104.

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Purification of Human Cofilin 1

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Skeletal actin [Spudich and Watt, 1971 (link)], WT yeast actin [Grintsevich et al., 2008 (link)], yeast cofilin [Grintsevich et al., 2008 (link)], FL drebrin [Sharma et al., 2010 (link)], and Drb1-300 construct [Grintsevich et al., 2010a (link)] were expressed and/or purified as described previously. Human cofilin 1 (hCof1) plasmid was a kind gift form Prof. E. M. De La Cruz. hCof1 expression in BL21(DE3)pLysS cells (4 hr, 37°C) was induced by 1 mM IPTG. HCof1 was purified employing ion exchange chromatography (strong cation exchanger, SP Sepharose FF, GE Healthcare) followed by gel-filtration on HiLoad 16/60 Superdex 75 (Amersham Biosciences) column.

Grintsevich E.E, & Reisler E. (2014). Drebrin Inhibits Cofilin-Induced Severing of F-Actin. Cytoskeleton (Hoboken, N.J.), 71(8), 472-483.

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Purification of Flag-Tagged SMARCAD1 from Sf9 Cells

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Flag-tagged SMARCAD1 was purified from baculovirus-infected Sf9 cells by three conventional chromatographic steps: SP-Sepharose FF (GE Healthcare), Q-Sepharose FF (GE Healthcare), and Toyopearl heparin (Tosoh). The peak fraction of the final Toyopearl heparin column in which Flag-tagged SMARCAD1 was expressed was immunopurified with ANTI-FLAG M2 agarose (Sigma-Aldrich). Flag-tagged SMARCAD1 was eluted with Flag peptides (Sigma-Aldrich) and dialyzed against 100 mM KCl buffer. Recombinant p300 was prepared as described previously.

Doiguchi M., Nakagawa T., Imamura Y., Yoneda M., Higashi M., Kubota K., Yamashita S., Asahara H., Iida M., Fujii S., Ikura T., Liu Z., Nandu T., Kraus W.L., Ueda H, & Ito T. (2016). SMARCAD1 is an ATP-dependent stimulator of nucleosomal H2A acetylation via CBP, resulting in transcriptional regulation. Scientific Reports, 6, 20179.

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Purification of Polyphenol Oxidase Enzymes

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The supernatant was loaded onto a cation exchange column (SP-Sepharose FF, GE Healthcare, length = 10 cm, diameter = 2.6 cm) and equilibrated with 20 mM sodium acetate, pH 4.5. Bound proteins were eluted with a linear gradient of sodium chloride (0–1 M) at a flow rate of 5 mL/min (see Fig. 1A). All collected fractions were tested photometrically for monophenolase and diphenolase activity. Fractions containing activity were pooled, ultra filtrated (size exclusion membrane of 10 kDa) and centrifuged (4000 rpm, 4 °C) to remove sodium chloride. The protein solution was then applied to a MonoS HR 5/50 Gl column (cation exchange column, GE Healthcare, length = 50 mm, diameter = 5 mm) and eluted with a linear gradient of sodium chloride (0–0.7 M) at a flow rate of 1 mL/min. Two forms were eluted at a conductivity of 13 and 16 mS/cm (see Fig. 1B), respectively. Fractions containing the first and the second eluted protein form were separately pooled and again loaded on the MonoS HR 5/50 Gl column under same conditions for removing further non-target proteins in a final polishing step (see Fig. 1C/D).

Zekiri F., Molitor C., Mauracher S.G., Michael C., Mayer R.L., Gerner C, & Rompel A. (2014). Purification and characterization of tyrosinase from walnut leaves (Juglans regia). Phytochemistry, 101(100), 5-15.

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Purification of Actin and Actin-Binding Proteins

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Rabbit skeletal actin (RSA) was purified from acetone powder^{27 (link)}. Recombinant Drosophila 5C actins (WT and M47L mutant) were purified from baculovirus-infected Sf9 cells^{12 (link)}. Recombinant human profilin-1 was expressed in E. coli and purified using poly-L-proline affinity column^{28 (link)}. Recombinant gelsolin segment-1 (6His-GS1) was expressed in E. coli and purified using metal chelate chromatography (Ni-NTA, Qiagen)^{26 (link)}. Human cofilin-1^{29 (link)} was expressed in E. coli BL21(DE3)pLysS cells and purified using SP Sepharose FF (GE Healthcare) followed by gel filtration on HiLoad 16/60 Superdex 75 (Amersham Biosciences). Drosophila Mical^redoxCH construct (referred to as Mical in this study) was expressed in E. coli ArcticExpress cells (Stratagene) and purified by two rounds of Ni²⁺ affinity chromatography followed by anion exchange on HiTrap Q-FF (GE Healthcare)^{30 (link)}. Acanthamoeba castellanii actin (AA) and mouse heterodimeric capping protein (CP) were a kind gift from Dr. Margot Quinlan (UCLA).

Grintsevich E.E., Ge P., Sawaya M.R., Yesilyurt H.G., Terman J.R., Zhou Z.H, & Reisler E. (2017). Catastrophic disassembly of actin filaments via Mical-mediated oxidation. Nature Communications, 8, 2183.

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Quaternization of Diblock Copolymer P2VP-b-PEO

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The diblock copolymer
poly(2-vinyl pyridine)₁₂₈-block-poly(ethylene
oxide)₄₇₇ (P2VP₁₂₈-b-PEO₄₇₇) (M_w/M_n = 1.1, M_n = 34.5 kg/mol) was
obtained from Polymer Source
Inc. After quaternization with iodomethane,^{23 (link)} the quaternization degree of this diblock copolymer was 73% (measured
by ¹H NMR) (Figure S1, Supporting
Information).^{24 (link)} All primers for gene modification
were obtained from Integrated DNA Technologies (IDT). DNA purification
kits (miniprep, PCR purification, and gel purification) were purchased
from Thermo Fisher Scientific. Lysogeny broth and agar medium for
the growth of Escherichia coli were
purchased from Duchefa Biochemie. Columns for enzyme purification
(SP-Sepharose FF, Q-Sepharose FF, Superdex 200, and Heparin Sepharose)
were obtained from GE Healthcare. Alexa Fluor 488 C5 maleimide obtained
from Thermo Fisher Scientific was used to fluorescently label the
CotA variants. To remove the unreacted label, a Biogel-P6DG gel filtration
column obtained from Bio-Rad was used. A Pierce bicinchoninic acid
(BCA) protein assay kit for the determination of total protein concentration
was obtained from Thermo Fisher Scientific. The substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS) for the activity assay was purchased from Sigma-Aldrich.

Kembaren R., Westphal A.H., Kamperman M., Kleijn J.M, & Borst J.W. (2022). Charged Polypeptide Tail Boosts the Salt Resistance of Enzyme-Containing Complex Coacervate Micelles. Biomacromolecules, 23(3), 1195-1204.

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Purification of T. thermophilus RNAP

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T. thermophilus RNAP core enzyme was prepared was prepared from T. thermophilus strain H8 (DSM 579; DSMZ), using polyethylenimine precipitation, ammonium sulfate precipitation, cation-exchange chromatography on SP Sepharose FF (GE Healthcare), anion-excahnge chromatography on Mono Q (GE Healthcare), and cation-exchange chromatography on Mono S (GE Healthcare); T. thermophilus σ^A was prepared from E. coli strain BL21(DE3) transformed with pET28a-Tt-σ^A, using immobilized-metal-ion affinity chromatography on Ni-NTA agarose (Qiagen) and anion-exchange chromatography on Mono Q (GE Healthcare); T. thermophilus RNAP core enzyme and T. thermophilus σ^A were combined to yield T. thermophilus RNAP σ^A holoenzyme; and T. thermophilus RNAP σ^A holoenzyme was further purified using size-exclusion chromatography on Superdex 200 (GE Healthcare), as in Zhang et al., 2014 (link).

Maffioli S.I., Zhang Y., Degen D., Carzaniga T., Gatto G.D., Serina S., Monciardini P., Mazzetti C., Guglierame P., Candiani G., Chiriac A.I., Facchetti G., Kaltofen P., Sahl H.G., Dehò G., Donadio S, & Ebright R.H. (2017). Antibacterial nucleoside-analog inhibitor of bacterial RNA polymerase. Cell, 169(7), 1240-1248.e23.

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Overexpression and Purification of GAPR-1

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Both wild-type (WT) and mutant GAPR-1 (UniProtKD ID: Q9H4G4) expression and purification have been described before [5 (link),19 (link)]. Briefly, the protein was overexpressed in Escherichia coli XL-1 Blue host cells and induced by 1 mM IPTG overnight at 18°C. After cell pelleting and homogenization with a high pressure homogenizer (Avestin, Mannheim, Germany), soluble proteins were collected by two-step centrifugation for 30 min at 14000×g followed by 30 min at 100000×g. GAPR-1 was purified by cation exchange chromatography using SP Sepharose FF (GE Healthcare, U.S.A.) eluted with a linear gradient of 0–400 mM NaCl in 25 mM Tris, pH 8.0. The purity of the isolated proteins was confirmed by SDS/PAGE and Coomassie Blue staining.

Sheng J., Olrichs N.K., Geerts W.J., Li X., Rehman A.U., Gadella B.M., Kaloyanova D.V, & Helms J.B. (2019). Zinc binding regulates amyloid-like aggregation of GAPR-1. Bioscience Reports, 39(2), BSR20182345.

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