Superdex 75 increase

Manufactured by GE Healthcare

Sourced in United Kingdom, United States

Superdex 75 Increase is a size exclusion chromatography medium designed for the separation and purification of biomolecules such as proteins, peptides, and other macromolecules. It is composed of highly cross-linked agarose beads that provide a porous structure for the size-based separation of molecules.

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Lab products found in correlation

Freestyle max 293 expression system, by Thermo Fisher Scientific (1 mentions) Sec uplc, by Waters Corporation (1 mentions) Human vegfr2, by R&D Systems (1 mentions) 293 expi cells, by Thermo Fisher Scientific (1 mentions) Akta explorer, by GE Healthcare (1 mentions) P7949, by Merck Group (1 mentions) Dithiothreitol (dtt), by neoFroxx (1 mentions) Odyssey fc, by LI COR (1 mentions) Dawn heleos, by Wyatt Technology (1 mentions) Astra software, by Wyatt Technology (1 mentions) Origin software, by OriginLab (1 mentions) His select ni2, by Merck Group (1 mentions) Hiload 16 60 superdex 200 pg gel filtration column, by GE Healthcare (1 mentions) Magicmedia, by Thermo Fisher Scientific (1 mentions) Vivaspin concentrator, by Sartorius (1 mentions) Ultimate 3000 hplc, by Thermo Fisher Scientific (1 mentions)

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12 protocols using superdex 75 increase

Isolation and Purification of VEGFR2-Binding Fab

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KD035 was identified by panning of Dyax FAB‐310 phagemid library on immobilized human VEGFR2 (R&D systems) followed by affinity maturation using a light chain shuffling method. The constructs carrying KD035 antigen‐binding fragment (Fab) domain were cloned into the mammalian expression vector pBh1 (Dyax) and transiently expressed in human 293 Expi cells (Invitrogen). Fabs were purified from cell culture supernatant by passing it several times through a protein A Sepharose HP column (GE Healthcare) using a peristaltic pump for a period of 8 h and eluting with 1 M Glycine pH 2.0. The eluate was further purified by size exclusion chromatography (SEC) using a Superdex 75 increase (GE Healthcare) column.
The variable domain amino acid sequence of IMC‐1121B (which shares the amino acid sequence of ramucirumab as described in US 8057791 B2/EP 1916001 A2) was reverse‐translated into DNA sequences and optimized by using Integrated DNA Technologies tools. The synthesized genes of 1121 light chain and heavy chain variable domains were cloned into a mammalian expression vector pBh1 (Dyax). The plasmid DNA containing 1121 gene was transfected and expressed by using the FreeStyle™ MAX 293 Expression System (ThermoFisher Scientific) according to the manufacture instruction. The supernatant was harvested and purified by protein A (GE) with at least 95% monomer detected by SEC‐UPLC (Waters).

Depetris R.S., Lu D., Polonskaya Z., Zhang Z., Luna X., Tankard A., Kolahi P., Drummond M., Williams C., Ebert M.C., Patel J.P, & Poyurovsky M.V. (2021). Functional antibody characterization via direct structural analysis and information‐driven protein–protein docking. Proteins, 90(4), 919-935.

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Protein Purification by Gel Filtration

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The chromatography column Superdex 75 Increase (GE HealthCare, Little Chalfont, UK) was equilibrated beforehand using gel filtration buffer (10 mM HEPES, 150 mM NaCl, 0.5 mM TECP, and 5% glycerol) at flow of 1 mL/min. Samples were loaded through loop of 5 mL using that column previously which was connected to chromatograph ÄktPrime^TM plus (GE HealthCare, Little Chalfont, UK) using gel filtration buffer as mobile phase. All proteins were collected in a range volume of 75–80 mL, analyzed by SDS-PAGE, concentrated up to 10 mg/mL, and then stored at −80 °C.

Dutra L.A., Heidenreich D., da Silva G.D., Man Chin C., Knapp S, & dos Santos J.L. (2017). Dietary Compound Resveratrol Is a Pan-BET Bromodomain Inhibitor. Nutrients, 9(11), 1172.

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Protein Purification via Gel Filtration

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The protein was loaded onto a Superdex 75 Increase gel-filtration column (GE HealthCare, 10/300 GL) in buffer containing 20 mM Hepes pH 7.5 and 250 mM NaCl. The resulting data were exported and plotted using GraphPad Prism.

Huang C., Xia M., Qiao H., Liu Z., Lin Y., Sun H., Yu B., Fang P, & Wang J. (2023). Tetramerization of upstream stimulating factor USF2 requires the elongated bent leucine zipper of the bHLH-LZ domain. The Journal of Biological Chemistry, 299(10), 105240.

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Structural Analysis of Irradiated TTR

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Size exclusion chromatography was performed using a Superdex 75 Increase analytical column (10/300 GL, GE Healthcare, Chicago, IL, USA) preequilibrated with Tris buffer. Prior to separation, samples containing TTR, riboflavin, and CaCl₂ in various combinations were irradiated at 23 °C for 30 min with light at an excitation wavelength of 445 nm using 2.0 nm slits. After irradiation, some samples were incubated overnight at 60 °C. Irradiated and nonirradiated samples were applied onto a column and analyzed in Tris buffer at a flow rate of 0.5 mL/min using Akta Explorer (GE Healthcare, Chicago, IL, USA).
For selected samples, 30 μL of the peak fractions was supplemented with four times concentrated sample buffer devoid or supplemented with 2.5% β-mercaptoethanol. Then, the samples were subjected to heat denaturation at 95 °C for 15 min or 20 min, except for the nonirradiated TTR sample, which was denatured at 85 °C for 30 min. All samples were loaded on 12% acrylamide gels containing SDS, run in a Laemmli buffer system and stained with Coomassie Brilliant Blue R250 and silver.

Wieczorek E., Wygralak Z., Kędracka-Krok S., Bezara P., Bystranowska D., Dobryszycki P, & Ożyhar A. (2022). Deep blue autofluorescence reflects the oxidation state of human transthyretin. Redox Biology, 56, 102434.

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Ark2C-Ubiquitin Interaction Analysis

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GST pull-down assays were performed by mixing GST-Ark2C proteins immobilised on Glutathione Sepharose^TM resin (GE Healthcare) with ubiquitin. Samples were made up to 200 μL in a buffer containing PBS pH 7.4, 0.2% Tween^® 20 (P7949, Sigma-Aldrich), and 2 mM DTT (1114GR005, BioFroxx), and incubated on a tube rotator at 4 °C for 30 min. Samples were then washed four times with PBS before being mixed with reducing 2× SDS sample buffer and resolved by SDS-PAGE. The pulldowns containing Cy3-labelled ubiquitin were imaged using an Odyssey^® Fc (LI-COR Biosciences) with a 600 nm filter prior to Coomassie staining.
Analytical SEC was performed using a 10/300 Superdex 75 increase (GE Healthcare) equilibrated with 20 mM Tris-HCl pH 7.5, 200 mM NaCl buffer. For each run, 100 µL of 200 µM of purified Ark2C variant, ubiquitin, or a mixture of the two proteins was resolved. For mixtures, samples were incubated overnight prior to separation. The recovered fractions were analysed by SDS-PAGE.

Paluda A., Middleton A.J., Rossig C., Mace P.D, & Day C.L. (2022). Ubiquitin and a charged loop regulate the ubiquitin E3 ligase activity of Ark2C. Nature Communications, 13, 1181.

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RNA Size-Exclusion Chromatography Analysis

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RNAs were analyzed on a Superdex 75 Increase (24 mL bed volume) size-exclusion column (GE Life Sciences) at 0.75 ml/min at 21 °C using a mobile phase composed of 20 mM Mops (pH 7.0), 150 mM KCl, 10 μM EDTA, and 5 mM MgCl₂. Absorbance was monitored at 260, 280, and 295 nm.

Passalacqua L.F., Starich M.R., Link K.A., Wu J., Knutson J.R., Tjandra N., Jaffrey S.R, & Ferré-D’Amaré A.R. (2023). Co-crystal structures of the fluorogenic aptamer Beetroot show that close homology may not predict similar RNA architecture. Nature Communications, 14, 2969.

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Purification of MST2 Kinase Variants

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25 μM of either MST2-K^WT, MST2-K^GC, or MST2-K^GN were run on a Superdex 75 Increase (10/300) GL (GE Healthcare) in 10mM Tris pH 8.0, 200mM NaCl, 5% glycerol, and 1mM TCEP.

Weingartner K.A., Tran T., Tripp K.W, & Kavran J.M. (2023). Dimerization and autophosphorylation of the MST family of kinases are controlled by the same set of residues. The Biochemical journal, 480(15), 1165-1182.

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Characterizing XPA-RPA Ternary Complexes

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In order to test the formation and stability of the ternary complexes, purified XPA_98–239 and RPA70AB proteins were mixed in an equimolar ratio with the DNA substrates at a final concentration of 50–100 μM. Analytical size-exclusion chromatography coupled to multi-angle light scattering (DAWN HELEOS, Wyatt Technology) (SEC-MALS) was used to monitor the system. A 24 ml Superdex 75 Increase analytical gel filtration column (GE Healthcare) was used with a flow rate of 0.5 ml/min in a buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol and 1 mM DTT. ASTRA software (Wyatt Technologies) was used for data processing, average molecular mass calculation and estimation of the peak monodispersity. To assess stoichiometry, fractions were collected and analyzed by SDS-PAGE.

Topolska-Woś A.M., Sugitani N., Cordoba J.J., Le Meur K.V., Le Meur R.A., Kim H.S., Yeo J.E., Rosenberg D., Hammel M., Schärer O.D, & Chazin W.J. (2020). A key interaction with RPA orients XPA in NER complexes. Nucleic Acids Research, 48(4), 2173-2188.

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Thermal Stability Analysis of DNA Sequences

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Three DNA^Myc sequences (IDT, Extended Data Fig. 5a) at 0.1 mM concentration in 20 mM cacodylic
acid-KOH (pH 7.2) and 20 mM or 150 mM KCl were heated at 95 °C for 2.5
min, placed on ice for 10 min, and warmed to 21 °C over 20 min. The DNAs
were analyzed by size-exclusion chromatography (Superdex 75 Increase, GE
Healthcare) in 20 mM cacodylic acid-KOH (pH 7.2) and either 20 mM or 150 mM KCl
(Extended Data Fig. 5b). For DSC, DNA
samples prepared in 20 mM cacodylic acid-KOH (pH 7.2) and 20 mM KCl were
degassed for 5–7 min prior to measurements (MicroCal VP-DSC differential
scanning calorimeter). Thermograms were acquired between 20–105 °C
at a scan rate of 0.5 °C min⁻¹ and at a constant
pressure of 24 p.s.i. Three to five heating and cooling cycles were collected at
least in duplicate for two independent preparations of each DNA sequence.
Thermograms were highly reproducible (Extended
Data Fig. 5c), and were analyzed with Origin software (OriginLab).
The reference ‘buffer versus buffer’ (20 mM cacodylic acid-KOH (pH
7.2) and 20 mM KCl) was subtracted from the sample data prior to curve-fitting
(Levenberg-Marquardt non-linear least-squares method) to determine
T_m and ΔH.

Chen M.C., Tippana R., Demeshkina N.A., Murat P., Balasubramanian S., Myong S, & Ferré-D’Amaré A.R. (2018). Structural basis of G-quadruplex unfolding by the DEAH/RHA helicase DHX36. Nature, 558(7710), 465-469.

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Characterization of hCD11bNb1 Binding to αMβ2 αI Domain

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For analysis of hCD11bNb1: α_Mβ₂–αI interaction, 40 μg α_Mβ₂ αI domain was incubated in the presence or the absence of a 1.5-fold molar excess of hCD11bNb1 in 20 mM Hepes (pH 7.5), and 150 mM NaCl. The mix was incubated for 30 min on ice and then applied to a 24 ml Superdex 75 increase (GE Healthcare) column equilibrated in 20 mM Hepes (pH 7.5) and 150 mM NaCl. For analysis of the inhibition, 170 μg αI domain and an equimolar amount of C3d were incubated for >5 min on ice in the presence or the absence of a twofold molar excess of hCD11bNb1 in a reaction buffer containing 20 mM Hepes (pH 7.5), 150 mM NaCl, and 2 mM MgCl₂. The mix was next applied to a 24 ml Superdex 75 increase column equilibrated in 20 mM Hepes (pH 7.5), 150 mM NaCl, and 2 mM MgCl₂.

Jensen R.K., Pedersen H., Lorentzen J., Laursen N.S., Vorup-Jensen T, & Andersen G.R. (2022). Structural insights into the function-modulating effects of nanobody binding to the integrin receptor αMβ2. The Journal of Biological Chemistry, 298(8), 102168.

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