Superdex 75 increase 10 300 column

Manufactured by GE Healthcare

The Superdex 75 Increase 10/300 column is a size exclusion chromatography column designed for the purification and analysis of proteins, peptides, and other biomolecules. The column features a matrix of cross-linked agarose and dextran, providing a separation range suitable for molecules between 3,000 and 70,000 daltons.

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Superdex 75 Increase 10/300 GL size exclusion column Superdex 75 Increase 10/300 GL column Superdex 75 Increase 10/300 GL gel filtration column Superdex 75 Increase 10/300 Superdex 75 10/300 column Superdex 75 Increase column Superdex 75 10/300 Superdex 75 Increase Superdex 75 column Superdex 75

12 protocols using superdex 75 increase 10 300 column

Analytical SEC of XIAP-BIR1 Monomer/Dimer

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To monitor XIAP‐BIR1 monomer/dimer equilibrium in the absence/presence of 2.5 mM 5 a or 5 b, analytical SEC experiments were performed at different protein concentrations. During the SEC experiments sample volumes of 50 μl, with XIAP‐BIR1 concentrated at 0.04 mM or 1 mM, were injected on a Superdex 75 Increase 10/300 column (GE Healthcare, volume=24 ml) attached to an ÄKTA Pure system in 20 mM Tris‐HCl pH 7.5, 200 mM NaCl, and 10 mM DTT. A 1 : 30 dilution ratio (injected sample volume/eluted peak volume) was estimated for all tested samples. Low molecular weight standards, from GE Healthcare, were used to calibrate the column.

Sorrentino L., Cossu F., Milani M., Malkoc B., Huang W., Tsay S., Ru Hwu J, & Mastrangelo E. (2019). Structure‐Activity Relationship of NF023 Derivatives Binding to XIAP‐BIR1. ChemistryOpen, 8(4), 476-482.

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Protein Binding Assay Protocol

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For each binding assay, proteins were incubated on ice for 15 min, at a 1:1.5 M ratio of ComR:Prx, with the exception of the DBD, where a higher molar ratio of DBD was used. Protein complexes were run over a HiLoad 16/600 superdex 75 column (GE Healthcare) or a Superdex75 increase 10/300 column (GE Healthcare) using an AKTA pure (GE Healthcare). All assays were performed with gel-filtration buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM β-ME).

Rutbeek N.R., Rezasoltani H., Patel T.R., Khajehpour M, & Prehna G. (2021). Molecular mechanism of quorum sensing inhibition in Streptococcus by the phage protein paratox. The Journal of Biological Chemistry, 297(3), 100992.

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Antibody Heavy and Light Chain Production

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The phCMV plasmids encoding heavy-chain or light-chains of each antibody were co-transfected into Expi-CHO cells using a standard protocol (Thermo Fisher Scientific). IgGs were purified with Protein A resin (Thermo Fisher Scientific). To generate Fab fragments of 25.10C, 19.7E or 37.7H, IgGs were digested with 5% with papain (Sigma-Aldrich) for 3 hours at 37 °C. The resulting Fabs were purified using anion exchange chromatography (Mono-Q 5/50 GL, GE Healthcare). Undigested IgG and F(ab’)2 were removed by size-exclusion chromatography (SEC, Superdex 75 increase 10/300 column, GE Healthcare).

Eis-Hübinger A.M., Däumer M., Matz B, & Schneweis K.E. (1999). Evaluation of Three Glycoprotein G2-Based Enzyme Immunoassays for Detection of Antibodies to Herpes Simplex Virus Type 2 in Human Sera. Journal of Clinical Microbiology, 37(5), 1242-1246.

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Purification of Strep-tagged GPCysR4 and scFvs

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The pMT-puro plasmids encoding Strep-tagged GPCysR4, Fabs (8.9F and 12.1F) or scFvs (8.9F, 12.1F and 37.2D) were used to establish stably expressing S2 cell lines. Stable cells were grown to a density of 1×10⁷ cells/mL and expression was induced by CuSO₄ at a final concentration of 500 μM. On day 5 after induction, proteins in the supernatants were purified with Strep-tactin resin (Qiagen) and the Strep-tags were removed by EKmax (Thermo Fisher Scientific) as needed. Further purification was performed with a Superdex 200 increase 10/300 column (for GPCysR4) or a Superdex 75 increase 10/300 column (for scFvs) (GE Healthcare).

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Oxidation Reaction Monitoring via SEC

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The progress of the oxidation reaction was monitored by SEC analysis of aliquotes (500 μL) of the incubation mixture taken at different time intervals. Gel filtration was performed with a Superdex 75 Increase 10/300 column (GE Healthcare, column volume 24 mL) on an AKTA purifier. The run was performed by using 20 mM ammonium acetate (pH 6.5) as running buffer, with a flow rate of 0.5 mL min⁻¹, and monitoring the protein elution by measuring absorbance at 280 nm. The protein fractions corresponding to peaks of reduced and oxidized Atox1 were individually collected, concentrated with Amicon Ultra centrifugal filters (3 kDa cutoff), and analyzed by mass spectrometry.

Nardella M.I., Rosato A., Belviso B.D., Caliandro R., Natile G, & Arnesano F. (2019). Oxidation of Human Copper Chaperone Atox1 and Disulfide Bond Cleavage by Cisplatin and Glutathione. International Journal of Molecular Sciences, 20(18), 4390.

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Multi-Angle Light Scattering Analysis of 4.1G-NuMA Fusion Proteins

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MALS analyses were performed on an AKTA™ pure HPLC system (GE Healthcare, USA) coupled to a DAWN HELEOS II eight-angle light-scattering and a refractive-index detector (Wyatt Technology, USA). 0.1 mL (8 mg/mL) of 4.1G-NuMA fusion proteins was passed over a Superdex 75 increase 10/300 column (GE Healthcare) in 20 mM Tris (pH 8.0), 150 mM NaCl, and 2 mM EDTA. Data were analyzed with the ASTRA (v7.0.1) software (Wyatt Technology) and graphed using Origin 2020.

Hu X., Xu Y., Wang C., Liu Y., Zhang L., Zhang J., Wang W., Chen Q, & Liu H. (2023). Combined prediction and design reveals the target recognition mechanism of an intrinsically disordered protein interaction domain. Proceedings of the National Academy of Sciences of the United States of America, 120(39), e2305603120.

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Size Exclusion Chromatography of Proteins

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Proteins were run on an AKTA pure with inline Superdex 75 Increase 10/300 column (GE Healthcare Life Sciences), miniDAWN TREOS, and Optilab T-rEX (Wyatt Technologies, Santa Barbara, CA). The column was equilibrated and run in SEC buffer, 500μL of 1-2mg/mL protein was loaded, and the column run at 0.5mL/min. Addition of 100mM maltose in the running buffer was used to determine the effect of laforin retention. The apparent molecular weight from SEC analysis was calculated by fitting the elution volume of the protein standards Bovine albumin, Ovalbumin, Carbonic Anhydrase and Lysozme. Light scattering data were processed using ASTRA (Wyatt Technologies). Molecular weight was determined by analyzing peaks at half height using Refractive Index (RI) as the concentration source. Further analysis and graphics were prepared using Prism (Graphpad Software, La Jolla, CA).

Sharma S., Vander Kooi C.D., Gentry M.S, & Vander Kooi C.W. (2018). Oligomerization and Carbohydrate Binding of Glucan Phosphatases. Analytical biochemistry, 563, 51-55.

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Purification and Characterization of AFF1/4-THD

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Wild type and mutants of AFF1/4-THD were purified according to the protocol aforementioned. Target proteins were applied on Superdex 75 10/300 increase column (GE) in 25 mM Tris pH 8.0, 150 mM NaCl, and 2 mM DTT. The concentration and injection volume were 6 mg/ml and 100 μl, respectively. The weight average molecular weight of target protein was calculated by Y = − 0.1725X + 5.2599 (R² = 0.99669, Y = lgMw, X = elution volume).

Tang D., Chen C., Liao G., Liu J., Liao B., Huang Q., Chen Q., Zhao J., Jiang H., Duan J., Huang J., Wang K., Wang J., Zhou C., Chu W., Li W., Sun B., Li Z., Dai L., Fu X., Cheng W., Xue Y, & Qi S. (2020). Structural and functional insight into the effect of AFF4 dimerization on activation of HIV-1 proviral transcription. Cell Discovery, 6, 7.

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Zn7MT-3 Metal-Protein Ratios

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Metal-to-protein ratios in the products of the reaction between Zn₇MT-3 and Cu(II) or Cu(I) and incubated at 25 °C up to 24 h were determined upon removing released metals through size-exclusion chromatography (SEC). Samples were injected on a Superdex 75 10/300 Increase column connected to anÄkta FPLC system (GE Healthcare) and eluted with 25 mM Tris-HCl pH 8.0, 50 mM NaCl.
Cu and Zn content in the eluted MT-3 samples was determined via ICP-MS (Agilent 7900) upon dilution in 1% HNO₃. The protein concentration was quantified photometrically in 0.1 M HCl using ε₂₂₀ = 53000 M⁻¹ cm⁻¹ (Cary 300 UV–vis spectrophotometer, Agilent), and metal-to-protein ratios were calculated.

Calvo J.S., Villones R.L., York N.J., Stefaniak E., Hamilton G.E., Stelling A.L., Bal W., Pierce B.S, & Meloni G. (2022). Evidence for a Long-Lived, Cu-Coupled and Oxygen-Inert Disulfide Radical Anion in the Assembly of Metallothionein-3 Cu(I)4-Thiolate Cluster. Journal of the American Chemical Society, 144(2), 709-722.

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Protein Characterization by SEC-MALS

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Size exclusion chromatography coupled with multiangle light scattering (SEC‐MALS) was performed on an Agilent1100 system at 22 °C. Hundred microlitres of the protein solution at 5 mg·mL⁻¹ was injected onto Superdex 75 10/300 Increase column connected to ÄKTA pure (GE Healthcare) equilibrated in 50 mm TRIS pH 7.8, 50 mm NaCl, 0.5 mm TCEP and run at 1.3 mL·min⁻¹. The chromatography system was connected to a multiangle light scattering detector DAWN HELEOS‐II (Wyatt Technology, Haverhill, UK) and Optilab T‐rEX refractometer (Wyatt Technology) connected in‐line. Data acquisition and processing were performed using astra software (Wyatt Technology).

Butryn A., Raza H., Rada H., Moraes I., Owens R.J, & Orville A.M. (2019). Molecular basis for GTP recognition by light‐activated guanylate cyclase RhGC. The Febs Journal, 287(13), 2797-2807.

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