Resource s column

Manufactured by GE Healthcare

Sourced in United States, United Kingdom

The Resource S column is a laboratory equipment product designed for column chromatography applications. It provides a platform for the separation and purification of various biomolecules, such as proteins, enzymes, and other macromolecules. The core function of the Resource S column is to facilitate the efficient separation and isolation of these target analytes from complex mixtures.

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Lab products found in correlation

Histrap hp column, by GE Healthcare (2 mentions) Hitrap, by Cytiva (2 mentions) Hitrap sp hp column, by GE Healthcare (2 mentions) Superdex 75 10 300 column, by GE Healthcare (1 mentions) Mini protein tetra cell system, by Bio-Rad (1 mentions) Ppiczαa vector, by Thermo Fisher Scientific (1 mentions) Akta pure, by GE Healthcare (1 mentions) M 110l microfluidizer, by Microfluidics (1 mentions) Sp sepharose, by GE Healthcare (1 mentions) Sep pak tc18 cartridge, by Waters Corporation (1 mentions) Rprotein a sepharose 4 fast flow, by GE Healthcare (1 mentions) Nhs ss peg4 biotin, by Thermo Fisher Scientific (1 mentions) Sephadex g 15, by GE Healthcare (1 mentions) Staphylococcus aureus v8 protease, by Fujifilm (1 mentions) Sepharose 4b, by GE Healthcare (1 mentions) Mucin from porcine stomach type 2, by Merck Group (1 mentions) Tskgel ods 120t, by Tosoh (1 mentions) Millex gv, by Merck Group (1 mentions) Cm sepharose fast flow, by GE Healthcare (1 mentions) Dc protein assay kit, by Bio-Rad (1 mentions)

Spelling variants of this product

Resource S (21 citations) Resource S cation-exchange column (4 citations) 6 mL Resource S column (2 citations) Resource S ion exchange column (2 citations)

30 protocols using resource s column

Purification and Fractionation of Crotamine

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Crude venom (70 mg) was dissolved in a 200 mM pH 3 ammonium formate buffer, centrifuged at 14,000 g for five minutes to remove insoluble material, and then fractionated using a Superdex 75 10/300 column (GE Healthcare, USA) equilibrated in the same buffer at a flow rate of 0.8 mL/minute. The absorbance of the eluate was monitored at 280 nm.
The crotamine fraction was pooled and refractionated using a 1 mL Resource S column (GE Healthcare, USA) equilibrated in 50 mM sodium phosphate buffer, pH 7.8 (buffer A). Buffer B was identical to buffer A, except that it was supplemented with 2.0 M NaCl. After an initial wash (5.0 mL) with 5 % buffer B, the protein was eluted with a linear gradient from 5 to 30 % buffer B. The fractions were pooled and then desalted by dialysis against water and lyophilized.

Oliveira K.C., Spencer P.J., Ferreira RS J.r, & Nascimento N. (2015). New insights into the structural characteristics of irradiated crotamine. The Journal of Venomous Animals and Toxins Including Tropical Diseases, 21, 14.

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PEGylation of Protein Analogs

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Thirtyfold molar excess of 20 kDa methoxypoly(ethylene glycol (mPEG)-maleimide (SunBio Inc.) was added to each analog solution (0.5 mg/mL) in 50 mM Tris buffer pH 8.0, incubated for 3 hours at room temperature. The reaction mixture was then diluted with 20 mM phosphate buffer (pH 7.0) and loaded onto a 6 mL pre-equilibrated cation exchange Resource S column (GE Healthcare, Piscataway, NJ, USA). After washing with 20 mM phosphate buffer (pH 7.0), bonded proteins were eluted using a gradient 20 mM phosphate buffer containing 500 mM NaCl adjusted to pH 7.0. Finally, PEGylated forms were analyzed by SDS-PAGE, Coomassie blue staining, using Mini-PROTEIN^® Tetra Cell system (Bio-Rad Laboratories).

Mirzaei H., Kazemi B., Bandehpour M., Shoari A., Asgary V., Ardestani M.S., Madadkar-Sobhani A, & Cohan R.A. (2016). Computational and nonglycosylated systems: a simpler approach for development of nanosized PEGylated proteins. Drug Design, Development and Therapy, 10, 1193-1200.

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Production and Purification of scFv-h3D6

Cited 1 time

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ScFv-h3D6 was produced as previously described [18 (link)]. Briefly, the scFv-h3D6 gene was cloned into the pPicZαA vector (Invitrogen, Carlsbad, CA, USA) and expressed in KM71H P. pastoris cells. Cells were grown until an OD₆₀₀ of 2.0 was reached. Then, methanol was supplemented every 24 h to induce protein expression. After 48 h of expression, cells were centrifuged at 3000× g and 4 °C for 10 min. ScFv-h3D6 was precipitated with ammonium sulfate. After centrifugation at 100,000× g and 4 °C for 1 h, the pellet was re-suspended in 10 mM Na₂HPO₄ (pH 6.5) and double dialyzed. Finally, cationic exchange chromatography was performed in a Resource S column coupled to a UP10 AKTA (GE Healthcare, Chicago, IL, USA). Pure scFv-h3D6 was dialyzed to PBS (pH 7.4) and stored at −20 °C until further use.

Roda A.R., Montoliu-Gaya L., Serra-Mir G, & Villegas S. (2020). Both Amyloid-β Peptide and Tau Protein Are Affected by an Anti-Amyloid-β Antibody Fragment in Elderly 3xTg-AD Mice. International Journal of Molecular Sciences, 21(18), 6630.

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Expression and Purification of Nanobody Nb6B9

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The expression and purification of Nb6B9 followed established protocols^{17 (link)}. In brief, the nanobody Nb6B9 was expressed in BL21-RIL E.coli cells and the cell pellets were lysed before clearing the lysate by centrifugation (75,600 g, 4 °C, 30 min). The cleared lysate was applied onto a HisTrap FF 5 mL Nickel affinity column on an AKTA Pure (GE Healthcare) pre-washed with equilibration buffer (20 mM Tris pH 8, 150 mM NaCl) and washed with the same buffer containing 15 mM imidazole before eluting the bound protein with 250 mM imidazole. The elution fractions were further used for cation exchange chromatography using a RESOURCE S column (GE Healthcare) pre-washed with equilibration buffer (50 mM Sodium Acetate pH 4.8, 75 mM NaCl). The nanobody was eluted from the RESOURCE S column with a linear NaCl gradient ranging from 75 mM to 1 M NaCl. The pure Nb6B9 was finally buffer exchanged into 10 mM Tris pH 8, 150 mM NaCl and concentrated to approximately 1 mM protein concentration.

Frei J.N., Broadhurst R.W., Bostock M.J., Solt A., Jones A.J., Gabriel F., Tandale A., Shrestha B, & Nietlispach D. (2020). Conformational plasticity of ligand-bound and ternary GPCR complexes studied by 19F NMR of the β1-adrenergic receptor. Nature Communications, 11, 669.

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Purification of Rpf2-Rrs1 Ribosome Biogenesis Complex

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Co-expression of Rpf2 and Rrs1-(His)₆ was carried out in E. coli BL21 (DE3) Rosetta 2 cells. Cells were grown in auto-induction medium (21 (link)) at 30°C for 8–10 hours under rigorous shaking. Expression of Se-Met labeled proteins was carried out as previously described (22 (link)).
Cell pellets were resuspended in lysis buffer (20 mM HEPES pH 7.5, 250 mM NaCl, 20 mM MgCl2, 20 mM KCl) and lysed with an M-110 L Microfluidizer (Microfluidics). The lysate was clarified by centrifugation and the supernatant loaded onto a HisTrap HP column (GE Healthcare). The column was washed with lysis buffer containing 40 mM imidazole. The protein was eluted in lysis buffer containing 500 mM imidazole. The protein was then loaded onto a Resource S column (GE Healthcare). The flow-through containing the protein complex was concentrated and subjected to size-exclusion chromatography (SEC) using a S75 26/60 column (GE Healthcare) in SEC buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 1% (v/v) glycerol). (His)₆-GST-ctRpL5 and (His)₆-GST-ctRpL11 proteins were purified by affinity chromatography in two steps using a HisTrap HP column (GE Healthcare) followed by a SP sepharose (GE Healthcare). (His)₆-ctRpf2, ctRrs1-(His)₆, the ctSyo1-RpL5-(His)₆-RpL11 complex and the ctSyo1-RpL5-(His)₆-RpL11–5S rRNA complex were purified as described (15 (link),16 (link)).

Kharde S., Calviño F.R., Gumiero A., Wild K, & Sinning I. (2015). The structure of Rpf2–Rrs1 explains its role in ribosome biogenesis. Nucleic Acids Research, 43(14), 7083-7095.

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Phosphopeptide Enrichment via SCX-TiO2

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Tryptic peptides were acidified with trifluoroacetic acid to 0.4% (vol/vol) and then desalted using 500 mg of tC18 Sep-Pak cartridges (500 mg; Waters, Dartford, UK) and lyophilized. Strong cation exchange (SCX) was performed on an Akta Purifier chromatography system using a 1-ml Resource S column (1 ml; GE Healthcare, Amersham, UK) with a flow rate of 1 ml/min and detection at 280 nm. Peptides were resuspended in buffer A (7 mM KH₂PO₄, pH 2.7, 30% [vol/vol] acetonitrile) and separated by a salt gradient consisting of 4 min of buffer A and a 4-min gradient to buffer A plus 350 mM KCl, followed by 3 min at buffer A plus 350 mM KCl. Fractions were collected throughout the run and combined into six or seven fractions. Combined fractions were lyophilized, desalted using 5-mg tC18 Sep-Pak cartridges) enriched for phosphopeptides using TiO₂ according to Wilson-Grady et al. (2008) (link), and lyophilized again before analysis.

Sugden C., Urbaniak M.D., Araki T, & Williams J.G. (2015). The Dictyostelium prestalk inducer differentiation-inducing factor-1 (DIF-1) triggers unexpectedly complex global phosphorylation changes. Molecular Biology of the Cell, 26(4), 805-820.

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Antibody Selection against Ci-VSD Mutants

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WT and R217E mutant of Ci-VSD-1-260 constructs were biotinylated through amine
coupling with NHS-SS-PEG4-Biotin (Thermo Scientific). Phage display selections were
performed using a synthetic antibody library built on the 4D5 Fab scaffold as described
previously (Rizk et al., 2011 (link)). Target protein
concentrations were serially adjusted using 100 nM in the first round and 10 nM and 5 nM
in subsequent rounds to ensure proper stringency. The conformation specific binders were
selected using a competitive selection strategy where 1 μM nonbiotinylated
competitor (such as Ci-VSD WT) was added to the phage library prior to the incubation with
actual selection target (such as Ci-VSD R217E). A single point competitive ELISA step used
to identify positive clones. Fabs were expressed in E. coli strain 55244 in phosphate
depleted media as described previously (Rizk et al.,
2011). Fabs were purified using automated two step protocol employing a custom
build AKTA purification system with 5ml rProteinA Sepharose 4 Fast Flow and 1ml Resource S
column (GE Healthcare) columns. Fractions were verified by SDS-PAGE, pooled and dialyzed
against PBS.

Li Q., Wanderling S., Paduch M., Medovoy D., Singharoy A., McGreevy R., Villalba-Galea C., Hulse R.E., Roux B., Schulten K., Kossiakoff A, & Perozo E. (2014). Structural Mechanism of Voltage-Dependent Gating in an Isolated Voltage-Sensing Domain. Nature structural & molecular biology, 21(3), 244-252.

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Isolation and Characterization of Pearl Shell Proteins

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Cultured large winged pearl shells (Pteria penguin, 6-years-old) and their larva were kindly provided by Amami South Sea & Mabe pearl Co. Ltd. (former Amami-branch, Tasaki & Co. Ltd.), Kagoshima, Japan. The mantle was collected and stored at −80°C until use. Resource S column, Sephadex G-15 and Sepharose 4B gels were purchased from GE Healthcare (NJ, USA). TSKgel sugar AXI, TSKgel ODS 120T, and TSKgel Amide 80 columns were from Tosoh (Tokyo, Japan). Trehalose-Sepharose 4B gel was prepared according to the method of Matsumoto et al. [29] . Mucin type I from bovine submaxillary glands and mucin type II from porcine stomach were purchased from Sigma Chemical (St. Louis, MO, USA). Achromobacter protease I and Staphylococcus aureus V8 protease were purchased from Wako Pure Chemical (Osaka, Japan). Glycopeptidase A was purchased from Seikagaku Kogyo (Tokyo, Japan). Trehalose was purchased from Hayashibara (Okayama, Japan). All the other reagents were of the purest grade commercially available.

Naganuma T., Hoshino W., Shikanai Y., Sato R., Liu K., Sato S., Muramoto K., Osada M., Yoshimi K, & Ogawa T. (2014). Novel Matrix Proteins of Pteria penguin Pearl Oyster Shell Nacre Homologous to the Jacalin-Related β-Prism Fold Lectins. PLoS ONE, 9(11), e112326.

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Purification and Separation of Polyubiquitin Chains

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Met1-linked di-ubiquitin was expressed as a linear fusion protein and purified by ion exchange chromatography and size exclusion chromatography. K11-, K48-, and K63-linked ubiquitin chains were enzymatically assembled using UBE2SΔC (K11), CDC34 (K48), and Ubc13/UBE2V1 (K63) as previously described (43 (link), 44 (link)). In brief, ubiquitin chains were generated by incubation of 1 μM E1, 25 μM of the respective E2 and 2 mM ubiquitin in reaction buffer (10 mM ATP, 40 mM Tris [pH 7.5], 10 mM MgCl₂, 1 mM DTT) for 18 h at RT. The reaction was stopped by 20-fold dilution in 50 mM sodium acetate (pH 4.5) and chains of different lengths were separated by cation exchange using a Resource S column (GE Healthcare). Elution of different chain lengths was achieved with a gradient from 0 to 600 mM NaCl.

Hermanns T., Woiwode I., Guerreiro R.F., Vogt R., Lammers M, & Hofmann K. (2020). An evolutionary approach to systematic discovery of novel deubiquitinases, applied to Legionella. Life Science Alliance, 3(9), e202000838.

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Purification of Antifungal Proteins

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PAF, PAF mutants and NFAP were purified from the supernatants of P. chrysogenum and P. digitatum. Shaking cultures of P. chrysogenum were first cleared from mycelia. The cell-free supernatant was ultra-filtered (Ultracell 30 kDa, Millipore, Billerica, MA, USA) and applied to a CM-Sepharose (Fast Flow, GE Healthcare Life Sciences, Little Chalfont, UK) column, equilibrated in phosphate buffer (10 mM NaPO₄, 25 mM NaCl, 0.15 mM EDTA, pH 6.6). The P. digitatum cell-free supernatant was dialyzed (2 K MWCO, Sigma-Aldrich, St Louis, MO, USA) against the phosphate buffer before applying to an AKTA Purifier system equipped with a 6 mL RESOURCE™ S column (GE Healthcare Life Sciences, Little Chalfont, UK), equilibrated in phosphate buffer. In all cases, the proteins were eluted applying 0.1-0.5 M NaCl. The protein containing fractions were pooled and dialyzed (3.5 K MWCO, ThermoFisher Scientific, Waltham, MA, USA) against ultra-pure ddH₂O and filter sterilized (0.22 µm, Millex-GV, PVDF, Millipore, Billerica, MA, USA). Protein concentrations were determined spectrophotometrically (A₂₈₀) considering the respective molar extinction coefficients and the purity was checked by SDS-PAGE using Coomassie blue staining.

Sonderegger C., Galgóczy L., Garrigues S., Fizil Á., Borics A., Manzanares P., Hegedüs N., Huber A., Marcos J.F., Batta G, & Marx F. (2016). A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses. Microbial Cell Factories, 15, 192.

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