Superdex 75 size exclusion chromatography, by GE Healthcare - Product details

1

Production and Purification of eOD-N276Kif

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eOD-N276Kif was produced in FreeStyleTM 293F (Invitrogen) suspension cultures by transient transfection using 293Fectin (Invitrogen) of a pHLSec plasmid containing gp120 with a C-terminal His₆ affinity tag in the presence of 25 μM kifunensine. Protein was harvested from the supernatant after 96 h and purified by affinity chromatography with a HisTrap column (GE) followed by Superdex 75 size exclusion chromatography (GE Healthcare) using an AKTA Express system (GE Healthcare).

Jardine J.G., Sok D., Julien J.P., Briney B., Sarkar A., Liang C.H., Scherer E.A., Henry Dunand C.J., Adachi Y., Diwanji D., Hsueh J., Jones M., Kalyuzhniy O., Kubitz M., Spencer S., Pauthner M., Saye-Francisco K.L., Sesterhenn F., Wilson P.C., Galloway D.M., Stanfield R.L., Wilson I.A., Burton D.R, & Schief W.R. (2016). Minimally Mutated HIV-1 Broadly Neutralizing Antibodies to Guide Reductionist Vaccine Design. PLoS Pathogens, 12(8), e1005815.

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2

Purification of Recombinant NTF2 Protein

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BL21(DE3) cells transfected with pET15b-NTF2⁷⁵ were inoculated into 5 mL of LB + Amp, and incubated at 37°C overnight. This starter culture was transferred to 2 L LB + Amp, grown at 37°C for ~5 h (until OD₆₀₀ ≈ 0.8), and then grown an additional 4 h at 27°C after inducing with 1 mM IPTG. Cells were resuspended in 20 mL of 20 mM Tris, 200 mM NaCl, 5 mM MgCl₂, 10 mM imidazole, pH 8.0 + PI, and then lysed as was done during the Ran purification. NTF2 was precipitated from the cleared lysate with saturated ammonium sulfate (at ~40-50% saturation), and recovered by centrifugation (4,000xg, 20 min, 4°C). The pellet was dissolved in 5 mL of 50 mM Tris, pH 8 over 1 h. The NTF2 solution was then purified using an HiPrep Q FF Anion Exchange Resin column (GE Healthcare Bioscience, #17-5190-01) with the dissolution buffer and a linear NaCl gradient, followed by Superdex 75 size exclusion chromatography (GE Healthcare Bioscience, #17-5174-01) in the dissolution buffer.

Chowdhury R., Sau A, & Musser S.M. (2022). Super-resolved 3D Tracking of Cargo Transport Through Nuclear Pore Complexes. Nature cell biology, 24(1), 112-122.

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3

Cloning and Purification of PerR Protein

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The full-length PerR of GAS serotype M1 A20 was cloned into a pET-21b vector between NdeI and XhoI restriction sites and transformed into E. coli BL21 (DE3). Expression of the recombinant 6xHIS-tagged PerR protein was induced by adding 0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside) to the culture when cells reached an O.D.₆₀₀ of 0.6, and further incubated at 25°C for 4 hr. Cells were centrifuged at 8,000 rpm for 20 min, resuspended in buffer A (20 mM Tris-HCl, 200 mM NaCl, pH 7.5) and disrupted by sonication on ice. Supernatant was loaded into a Ni-NTA column (GE Healthcare) and unbound proteins were washed away with 60 mM imidazole in buffer A. PerR proteins were eluted with 300 mM imidazole in buffer A. Fractions containing PerR proteins were pooled and further purified by Superdex™ 75 size exclusion chromatography (GE Healthcare). Subsequently, proteins were dialyzed in buffer B (20 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 5% glycerol, and 3 mM DTT, pH 7.5). Purified proteins were stored at 4°C for further use. The PerR mutant plasmids were constructed by the method of Dpn I-dependent site-directly mutagenesis (SDM) or overlap extension PCR (OE-PCR) using wild-type PerR as a template. All primers used for construction of PerR and PerR mutant plasmids are listed in Table S1. Overexpression and purification of PerR mutant proteins were performed as described above.

Lin C.S., Chao S.Y., Hammel M., Nix J.C., Tseng H.L., Tsou C.C., Fei C.H., Chiou H.S., Jeng U.S., Lin Y.S., Chuang W.J., Wu J.J, & Wang S. (2014). Distinct Structural Features of the Peroxide Response Regulator from Group A Streptococcus Drive DNA Binding. PLoS ONE, 9(2), e89027.

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4

Purification of E. coli-Expressed Polη

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Polη was expressed and purified from E. coli with minor modifications of the method described previously [32 (link)]. Briefly, Polη was overexpressed in E. coli BL21(DE3) cells, and cultures were grown in Luria-Bertani medium at 37 °C until reaching the OD₆₀₀ of 0.7, and the cells were induced by adding 0.2 mM isopropyl β-D-α-thiogalactopyranoside. After incubating for 18 hours at 20 °C, the pelleted cells (6,000 RPM for 30 min) were resuspended in Ni–NTA column binding buffer A (50 mM sodium phosphate, pH 7.8, 500 mM NaCl and 10% glycerol) supplemented with 1 mg/ml lysozyme, 0.25% NP-40, 0.25% Triton X-100, and 0.25 mM phenylmethylsulfonyl fluoride (PMSF). After sonication for 90 seconds, the lysate was centrifuged at 15,000 g at 4 °C for 20 min. The supernatant was then filtered through 0.22 μm filter and further purified through Ni–NTA column (GE Healthcare). The elution fractions were pooled and further purified using the Heparin HiTrap column (GE Healthcare) followed by Superdex-75 size exclusion chromatography (GE Healthcare). The purity of the final product was confirmed by SDS-PAGE gel. The purified protein was concentrated, flash-frozen in liquid nitrogen, and stored at −80 °C for the future use.

Jung H., Hawkins M.A, & Lee S. (2020). Structural insights into the bypass of the major deaminated purines by translesion synthesis DNA polymerase. The Biochemical journal, 477(24), 4797-4810.

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5

Isotopically Labeled Protein NMR Characterization

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For NMR samples, bacteria were grown in M9 medium. Uniformly labeled BAK 15–186 was produced using M9 medium containing 0.5 g L⁻¹ 99% ¹⁵N-ammonium sulfate and 2.5 g L⁻¹ 99% ¹³C-glucose as the sole nitrogen and carbon sources, respectively. After Ni²⁺-NTA column purification, the labeled protein was further purified using Superdex-75 size exclusion chromatography (GE) and dialyzed against 20 mM NaH₂PO₄ (pH 6.7) containing 100 mM NaCl, 1 mM DTT, and 2 mM EDTA.
All NMR spectra were recorded at 303 K on a Bruker DMX850 spectrometer with Topspin software. To assign backbone residues of BAK 15–186, a set of standard 2D and 3D spectra was recorded, including ¹H-¹⁵N-HSQC (heteronuclear single-quantum correlation), HNCACB, CACB(CO)NH, HNCA, HN(CA)CO. All NMR data were processed with NMRPipe^{44 (link)} and analyzed with Sparky 3 software^{45 (link)}.

Ye K., Meng W.X., Sun H., Wu B., Chen M., Pang Y.P., Gao J., Wang H., Wang J., Kaufmann S.H, & Dai H. (2020). Characterization of an alternative BAK-binding site for BH3 peptides. Nature Communications, 11, 3301.

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6

Purification of RhoA-related Proteins

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RHOA proteins, p190RhoGAP domain, ECT2-PH-DH domain, ROCK-RBD, mDia-RBD and Rhotekin-RBD were expressed in E. coli BL21 (DE3) Rosetta2 cells in Terrific Broth (TB) medium by overnight expression at 18°C following induction with 250 μM isopropyl-β-D-1-thiogalactopyranoside at an OD₆₀₀ of around 0.6 at 37°C, as previously described (23 (link)). ROCK-RBD, mDia-RBD, Rhotekin-RBD and p190RhoGAP were purified as GST-fusion proteins at 4°C using glutathione sepharose 4B chromatography (GE Life Sciences) accordingly manufacture’s guidelines, followed by cleavage of the GST-tag (only for p190RhoGAP) and Superdex75 size exclusion chromatography (GE Life Sciences) as previously described (23 (link)). ECT2 was purified as a His-fusion protein at 4°C using HisTRAP nickel Sepharose chromatography (GE Life Sciences) accordingly standard protocols, followed by cleavage of the His-tag and size exclusion chromatography (23 (link)). RHOA proteins were purified either as GST- or His-tagged protein, followed by tag cleavage and size exclusion chromatography. Fractions were analyzed by SDS-PAGE, fractions with greater than 90% purity were pooled, aliquoted, flash frozen in liquid nitrogen and stored at −80°C. Proteins were stored in 50 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol and 5 mM β-mercaptoethanol. Buffer for RHOA contained in addition 5 mM MgCl₂.

Nüsslein K, & Tiedje J.M. (1999). Soil Bacterial Community Shift Correlated with Change from Forest to Pasture Vegetation in a Tropical Soil. Applied and Environmental Microbiology, 65(8), 3622-3626.

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7

Production and Purification of Avi-tagged and Biotinylated eOD Monomers

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Avi-tagged and biotinylated versions of eOD-GT8 monomer (21 (link), 39 (link)) and eOD-GT8-KO11 monomer (different than the original eOD-GT8-KO) (43 , 46 (link)), were produced by a cGLP lab under contract at Scripps Research. These proteins were produced as previously described (13 (link)) in FreeStyle 293F (Invitrogen) suspension cultures by transient transfection using 293Fectin (Invitrogen) of a pHLSec plasmid containing mammalian codon-optimized eOD with a C-terminal His6x affinity tag followed by an Avi tag. Protein was harvested from the supernatant after five days and purified by affinity chromatography with a HIS-TRAP column (GE Healthcare) followed by Superdex 75 size exclusion chromatography (GE Healthcare) using an AKTA chromatography system (GE Healthcare). Biotinylation was accomplished using BirA (Avidity), and the level of biotinylation was estimated by PAGE.

Leggat D.J., Cohen K.W., Willis J.R., Fulp W.J., deCamp A.C., Kalyuzhniy O., Cottrell C.A., Menis S., Finak G., Ballweber-Fleming L., Srikanth A., Plyler J.R., Schiffner T., Liguori A., Rahaman F., Lombardo A., Philiponis V., Whaley R.E., Seese A., Brand J., Ruppel A.M., Hoyland W., Yates N.L., Williams L.D., Greene K., Gao H., Mahoney C.R., Corcoran M.M., Cagigi A., Taylor A., Brown D.M., Ambrozak D.R., Sincomb T., Hu X., Tingle R., Georgeson E., Eskandarzadeh S., Alavi N., Lu D., Mullen T.M., Kubitz M., Groschel B., Maenza J., Kolokythas O., Khati N., Bethony J., Crotty S., Roederer M., Karlsson Hedestam G.B., Tomaras G.D., Montefiori D., Diemert D., Koup R.A., Laufer D.S., McElrath M.J., McDermott A.B, & Schief W.R. (2022). Vaccination induces HIV broadly neutralizing antibody precursors in humans. Science (New York, N.Y.), 378(6623), eadd6502.

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8

Purification of Recombinant NTF2 Protein

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BL21(DE3) cells transfected with pET15b-NTF2⁷⁵ were inoculated into 5 mL of LB + Amp, and incubated at 37°C overnight. This starter culture was transferred to 2 L LB + Amp, grown at 37°C for ~5 h (until OD₆₀₀ ≈ 0.8), and then grown an additional 4 h at 27°C after inducing with 1 mM IPTG. Cells were resuspended in 20 mL of 20 mM Tris, 200 mM NaCl, 5 mM MgCl₂, 10 mM imidazole, pH 8.0 + PI, and then lysed as was done during the Ran purification. NTF2 was precipitated from the cleared lysate with saturated ammonium sulfate (at ~40-50% saturation), and recovered by centrifugation (4,000xg, 20 min, 4°C). The pellet was dissolved in 5 mL of 50 mM Tris, pH 8 over 1 h. The NTF2 solution was then purified using an HiPrep Q FF Anion Exchange Resin column (GE Healthcare Bioscience, #17-5190-01) with the dissolution buffer and a linear NaCl gradient, followed by Superdex 75 size exclusion chromatography (GE Healthcare Bioscience, #17-5174-01) in the dissolution buffer.

Chowdhury R., Sau A, & Musser S.M. (2022). Super-resolved 3D Tracking of Cargo Transport Through Nuclear Pore Complexes. Nature cell biology, 24(1), 112-122.

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9

Purification of LaG9(S151C) Protein

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BL21(DE3) cells transfected with pET21b-LaG9(S151C) were inoculated into 5 mL of LB + Amp, and cultured overnight at 37°C. The starter culture was transferred into 1 L of LB + Amp, which was grown at 37°C until the OD₆₀₀ was ~0.8, and then induced with 0.1 mM IPTG for 20 h at 20°C. The cells were resuspended in 10 mL ice cold 20 mM Tris, 0.5 mM EDTA, 500 mM sucrose, 10 mM βME, 0.3 mM MgSO₄, pH 8.0 + PI, and then lysed as was done during the Ran purification. The cleared lysate was incubated with 0.5 mL Ni-NTA resin for 30 min at 4°C, and the suspension was then transferred to a gravity column. The resin was washed with 15 mL of 20 mM Tris, 900 mM NaCl, 10 mM βME, 0.1% TX-100, pH 8.0, and then 15 mL of 20 mM Tris, 150 mM NaCl, 10 mM imidazole, pH 8.0. Fractions (0.5 mL) were eluted with 20 mM Tris, 150 mM NaCl, 250 mM imidazole, pH 8.0. The most concentrated fraction was further purified by Superdex 75 size exclusion chromatography (GE Healthcare Bioscience, #17-5174-01) using 20 mM Hepes, 150 mM NaCl, pH 7.3.

Chowdhury R., Sau A, & Musser S.M. (2022). Super-resolved 3D Tracking of Cargo Transport Through Nuclear Pore Complexes. Nature cell biology, 24(1), 112-122.

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10

Purification of LaG9(S151C) Protein

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BL21(DE3) cells transfected with pET21b-LaG9(S151C) were inoculated into 5 mL of LB + Amp, and cultured overnight at 37°C. The starter culture was transferred into 1 L of LB + Amp, which was grown at 37°C until the OD₆₀₀ was ~0.8, and then induced with 0.1 mM IPTG for 20 h at 20°C. The cells were resuspended in 10 mL ice cold 20 mM Tris, 0.5 mM EDTA, 500 mM sucrose, 10 mM βME, 0.3 mM MgSO₄, pH 8.0 + PI, and then lysed as was done during the Ran purification. The cleared lysate was incubated with 0.5 mL Ni-NTA resin for 30 min at 4°C, and the suspension was then transferred to a gravity column. The resin was washed with 15 mL of 20 mM Tris, 900 mM NaCl, 10 mM βME, 0.1% TX-100, pH 8.0, and then 15 mL of 20 mM Tris, 150 mM NaCl, 10 mM imidazole, pH 8.0. Fractions (0.5 mL) were eluted with 20 mM Tris, 150 mM NaCl, 250 mM imidazole, pH 8.0. The most concentrated fraction was further purified by Superdex 75 size exclusion chromatography (GE Healthcare Bioscience, #17-5174-01) using 20 mM Hepes, 150 mM NaCl, pH 7.3.

Chowdhury R., Sau A, & Musser S.M. (2022). Super-resolved 3D Tracking of Cargo Transport Through Nuclear Pore Complexes. Nature cell biology, 24(1), 112-122.

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Superdex 75 size exclusion chromatography

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10 protocols using superdex 75 size exclusion chromatography

Production and Purification of eOD-N276Kif

Purification of Recombinant NTF2 Protein

Cloning and Purification of PerR Protein

Purification of E. coli-Expressed Polη

Isotopically Labeled Protein NMR Characterization

Purification of RhoA-related Proteins

Production and Purification of Avi-tagged and Biotinylated eOD Monomers

Purification of Recombinant NTF2 Protein

Purification of LaG9(S151C) Protein

Purification of LaG9(S151C) Protein

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