Superdex 75 increase column

Manufactured by GE Healthcare

Sourced in United States

The Superdex 75 Increase column is a pre-packed size exclusion chromatography column designed for the purification and analysis of proteins, peptides, and other biomolecules. The column features a porous agarose-based matrix that separates analytes based on their molecular size and shape.

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Lab products found in correlation

Pcdna3.1 vector, by GenScript (1 mentions) Dylight800 maleimide, by Thermo Fisher Scientific (1 mentions) Sp sepharose fast flow, by GE Healthcare (1 mentions) Alexa 647, by Thermo Fisher Scientific (1 mentions) Sodium azide, by Merck Group (1 mentions) Akta upc 10 fplc system, by GE Healthcare (1 mentions) Hispur cobalt resin, by Thermo Fisher Scientific (1 mentions) Sodium chloride (nacl), by Merck Group (1 mentions) Imidazole, by Merck Group (1 mentions) Fluostar plate reader, by BMG Labtech (1 mentions) 5 5 dithiobis 2 nitrobenzoic acid, by Merck Group (1 mentions) Centrifugal filter, by Merck Group (1 mentions) Quikchange lightning site directed mutagenesis kit, by Agilent Technologies (1 mentions) Resource q, by GE Healthcare (1 mentions) Complete edta free protease inhibitor, by Roche (1 mentions) Strep tactin superflow plus column, by Qiagen (1 mentions)

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10 protocols using superdex 75 increase column

Protein purification by size-exclusion

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Size-exclusion chromatography was performed on a Superdex-75 Increase column (GE), on an AKTA Purifier 10 workstation (GE), with equilibration of the column with Tris (50 mM) buffer of pH 7.4 containing 150 mM KCl, at room temperature. All protein loading was carried out using 500 μl protein of 110 μM concentration. Monitoring of the elution volume(s) of protein(s) was done through the measurement of the 280 nm UV absorption of the eluent.

Gupta A., Joshi A., Arora K., Mukhopadhyay S, & Guptasarma P. (2023). The bacterial nucleoid-associated proteins, HU and Dps, condense DNA into context-dependent biphasic or multiphasic complex coacervates. The Journal of Biological Chemistry, 299(5), 104637.

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Expressing and Purifying scFv Antibody Fragments

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The scFv sequences were cloned into pcDNA3.1 vector with an N-terminal IL2 secretion sequence and a C-terminal His tag (GenScript, Piscataway, NJ). Expression was done using a protocol adapted from Ref. 66 (link). Briefly, 1 mg of purified plasmid DNA was transfected with polyethyleneimine at a ratio of 1:3 into 1 liter of Freestyle293 cells at a density of 2–2.5 × 10⁶ cells/ml and incubated at 37 °C for 7 days. The medium was harvested via centrifugation and filtered, and the scFv was purified via stepwise affinity and size-exclusion chromatography on a Superdex 75 Increase column (GE Healthcare). Alternatively, recombinant scFvs containing a C-terminal FLAG^TM tag (DYKDDDDK) were produced in E. coli by AxioMx (AxioMx, Abcam, Cambridge, UK).

Miller M.S., Douglass J., Hwang M.S., Skora A.D., Murphy M., Papadopoulos N., Kinzler K.W., Vogelstein B., Zhou S, & Gabelli S.B. (2019). An engineered antibody fragment targeting mutant β-catenin via major histocompatibility complex I neoantigen presentation. The Journal of Biological Chemistry, 294(50), 19322-19334.

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Expression and Purification of Fluorescent Ubiquitin

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Recombinant human ubiquitin with an N-terminal Met-Gly-Pro-Leu-Cys-Gly-Ser overhang was expressed at 37°C in BL21(DE3) cells using autoinduction medium. Following an established ubiquitin purification procedure (Ecker et al., 1987 (link)), the expression pellet was resuspended in 50 mM ammonium acetate pH 4.5 and the cells opened by sonication (Branson Digital Sonifier 450, Marshall Scientific). The lysate was centrifugated for 30 min at 40,000 × g, after which the supernatant was heat-denatured for 10 min at 70°C and re-centrifugated. Ubiquitin was finally purified by IEX using SP Sepharose Fast Flow (GE Healthcare) and size exclusion chromatography (SEC) using a Superdex 75 Increase column (GE Healthcare). The protein was concentrated by Vivaspin ultrafiltration (5 kDa cutoff, Sigma-Aldrich #Z614580) to 10 mg/mL. Purified ubiquitin was fluorescently labeled with DyLight800-Maleimide (Thermo Fisher Scientific #46621) and re-purified by IEX as before, and SEC using 5 mM phosphate buffer, pH 7.5. The final ubiquitin sample was concentrated to 5 mg/mL, flash-frozen in liquid nitrogen, and stored at –80°C.

Ahel J., Lehner A., Vogel A., Schleiffer A., Meinhart A., Haselbach D, & Clausen T. (2020). Moyamoya disease factor RNF213 is a giant E3 ligase with a dynein-like core and a distinct ubiquitin-transfer mechanism. eLife, 9, e56185.

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Fluorescent Labeling of NbSyn87 Nanobody

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NbSyn87 protein was obtained from a custom production service offered by NanoTag Biotechnologies GmbH (Göttingen, Germany), and the nanobody was equipped with one ectopic cysteine at its C-terminal. 1 mg of pure NbSyn87 was reduced by adding TCEP (Sigma-Aldrich) to a final concentration of 5 mM for 1 h. The reduced sample was then desalted using gravity columns Nap10 (GE Healthcare Life Sciences) in nitrogen bubbled PBS (pH 7.4) and immediately added to five molar excess of maleimide-functionalized Alexa647 (Thermo Scientific) for 1 h. Excess of free dye was separated from the conjugated nanobody with an Äkta HPLC equipped with a Superdex 75 increase column (GE Healthcare Life Sciences).

Gerdes C., Waal N., Offner T., Fornasiero E.F., Wender N., Verbarg H., Manzini I., Trenkwalder C., Mollenhauer B., Strohäker T., Zweckstetter M., Becker S., Rizzoli S.O., Basmanav F.B, & Opazo F. (2020). A nanobody-based fluorescent reporter reveals human α-synuclein in the cell cytosol. Nature Communications, 11, 2729.

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Cobalt Affinity Purification Protocol

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After filtration by using a 0.22 μm filter cup (Thermo), the supernatant was mixed with the binding buffer (500 mM Tris-HCl (Sigma, Aldrich, St. Louis, MO, USA), 1.5 M sodium chloride (Sigma), and 50 mM imidazole (Sigma), pH 7.8) with a ratio of 10:1. The HisPur™ Cobalt Resin (Thermo), which was pre-washed with sterile water and TBA buffer (50 mM Tris-HCl (Sigma), 150 mM sodium chloride (Sigma), 0.02% sodium azide (Sigma), pH 7.6) was added to the supernatant in the binding buffer in a ratio of 1:100. After overnight incubation at 4 °C with regular stirring, the cobalt resin was collected by the Glass Econo-Column^® Column (Bio-Rad, Hercules, CA, USA), and washed with the 5-fold resin volume of wash buffer (20 mM Tris-HCl (Sigma), 300 mM sodium chloride (Sigma), and 10 mM imidazole (Sigma), pH 7.6). The target protein was eluted by using the elution buffer (20 mM Tris-HCl (Sigma), 300 mM sodium chloride (Sigma), and 150 mM imidazole (Sigma), pH 7.8). The eluent was concentrated to 0.5 mL for further purification in PBS (Sigma) by using a Superdex™ 75 Increase column (GE Healthcare, Chicago, IL, USA) coupled to an AKTA UPC10 FPLC system (GE Healthcare).

Chang C.Y., Wang Y.S., Wu J.F., Yang T.J., Chang Y.C., Chae C., Chang H.W, & Hsu S.T. (2021). Generation and Characterization of a Spike Glycoprotein Domain A-Specific Neutralizing Single-Chain Variable Fragment against Porcine Epidemic Diarrhea Virus. Vaccines, 9(8), 833.

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Amyloid-beta Aggregation Kinetics Assay

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Aβ42(M1-42) was expressed in E. coli BL21 Gold (DE3) with a methionine at the N-terminus (elsewhere referred to as Aβ42) and the inclusion body was extracted and purified using ion-exchange (IEX) and size-exclusion chromatography (SEC). The target protein was eluted in buffer with 125 mM NaCl during the IEX from the DE23 anion exchange resin and followed by two rounds of SEC using Superdex75 increase column (GE Healthcare). The protein was purified and diluted to the required concentration in 20 mM sodium phosphate buffer at pH 8.0 with 0.2 mM of EDTA. Preformed fibrils (seeds) were prepared by aggregating 5 µM monomeric at 37 °C and diluted to the needed concentration. SG2 was dissolved in MQ water at a stock concentration of 5 mM. The molecule was added to Aβ42 to reach a final concentration of 5 µM to 20 µM. 20 µM thioflavin T (ThT) was added to the solution to serve as a fluorescence probe for the aggregation. The kinetics of Aβ42 aggregation was recorded in the Fluostar plate reader (BMG Labtech) with an excitation wavelength at 440 nm and emission wavelength at 480 nm.

Runfola M., Perni M., Yang X., Marchese M., Bacci A., Mero S., Santorelli F.M., Polini B., Chiellini G., Giuliani D., Vilella A., Bodria M., Daini E., Vandini E., Rudge S., Gul S., Wakelam M.O., Vendruscolo M, & Rapposelli S. (2021). Identification of a Thyroid Hormone Derivative as a Pleiotropic Agent for the Treatment of Alzheimer’s Disease. Pharmaceuticals, 14(12), 1330.

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Analyzing Copper-binding Cysteine Mutants

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DTT-treated Cys65Ser PmoD was exchanged into reductant-free buffer prior to the addition of two molar equivalents of CuSO₄. Excess copper was then removed by desalting on a PD10 column into 4 mL copper-free buffer. A 1 mL aliquot of this sample was immediately run on a Superdex 75 Increase column (GE). A second 1 mL aliquot was treated with 50 mM EDTA, pH 8 and incubated on ice for 3 hr prior to running on the Superdex 75 Increase column. The remaining sample was incubated on ice at 4 °C for 144 hr to allow the Cu_A site to decay and subjected to size exclusion chromatography and EDTA treatment as described for the earlier time points. The peak fractions from each column were pooled and concentrated using 10 kDa molecular weight cut-off centrifugal concentrators (Millipore). Approximately 60 μg of each sample was run on a denaturing gel in either the absence or presence of β-mercaptoethanol, and ICP-OES was used to confirm that the EDTA treatment effectively removed copper. The Cys41Ser sample was prepared as described for the Cys65Ser sample and run on a HiLoad 16/600 Superdex 75 column in the absence of reducing agents.

Ross M.O., Fisher O.S., Morgada M.N., Krzyaniak M.D., Wasielewski M.R., Vila A.J., Hoffman B.M, & Rosenzweig A.C. (2019). Formation and electronic structure of an atypical CuA site. Journal of the American Chemical Society, 141(11), 4678-4686.

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Ubiquitin Conjugating Enzyme Complex Formation

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The same approach was used to generate complexes for UBE2D2 and UBE2D3, referred to collectively as UBE2D. UBE2D C21I C107A C111D was purified from size exclusion chromatography (see above) and then immediately used without freezing. After SEC, the protein was concentrated (Amicon, EMD Milipore) to 600 μM. 2 × 100 μL of protein were separately desalted (2 × Zeba, 0.5 mL column, 7K MWCO, ThermoFisher) to 20 mM HEPES, 250 mM NaCl, 5 mM EDTA pH 7.0. Elutions were combined and immediately added together to 34 μL 10 mM 5,5’-dithiobis-(2-Nitrobenzoic acid) (SigmaAldrich, dissolved in 50 mM NaPO₄ pH 7.5) and mixed by pipetting before incubating at room temperature for 30 min. The solution was then desalted (2 × Zeba, 0.5 mL column, 7K MWCO, ThermoFisher) to 20 mM HEPES, 250 mM NaCl, 5 mM EDTA pH 7.0 at the same time that UB(1–75)_Cys_IκBα (500 μL at 100 μM) was desalted (1 × Zeba, 2 mL column, 7K MWCO, ThermoFisher) to the same buffer. The UBE2D and UB components were then immediately combined and incubated at room temperature for 30 min, at which point, the sample was loaded to a Superdex 75 Increase column (GE Healthcare) equilibrated with 20 mM HEPES, 250 mM NaCl, 5 mM EDTA pH 7.0.

Baek K., Krist D.T., Prabu J.R., Hill S., Klügel M., Neumaier L.M., von Gronau S., Kleiger G, & Schulman B.A. (2020). NEDD8 nucleates a multivalent cullin-RING-UBE2D ubiquitin ligation assembly. Nature, 578(7795), 461-466.

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Labeling of β2GPI Reduced Thiols

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All reaction steps were performed under argon atmosphere at 25 °C. 50 µM recombinant human thioredoxin-1 (TRX-1) was reduced by 50 µM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) in a total volume of 100 µL Hepes-buffered saline (HBS; 20 mM Hepes, 140 mM NaCl, pH 7.4) for 1 h with mild shaking. 900 µL HBS buffer and 0.2 µM β2GPI were added and incubated for 1 h at with mild shaking. 15 µM MPB (N^a-(3-maleimidylpropionyl)biocytin; Thermo Fisher) were added for 15 min with mild shaking to label free thiols. Finally, 200 µM reduced glutathione (GSH; Thermo Fisher) was used to quench unbound MPB (15 min). For purification, the reaction mixture was first volume-reduced by a 10 kDa cut-off centrifugal filter unit (Merck) at 2500 × g, followed by size exclusion chromatography (SEC) with a Superdex 75 Increase column (GE Healthcare) with a flow rate of 0.25 mL/min in HBS buffer. β2GPI main peak SEC fractions were pooled and concentrated using centrifugal filters with a cut-off of 10 kDa (Merck) at 4000×g.

Buchholz I., McDonnell T., Nestler P., Tharad S., Kulke M., Radziszewska A., Ripoll V.M., Schmidt F., Hammer E., Toca-Herrera J.L., Rahman A, & Delcea M. (2021). Specific domain V reduction of beta-2-glycoprotein I induces protein flexibility and alters pathogenic antibody binding. Scientific Reports, 11, 4542.

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Purification of Mad1 C-Terminal Domain

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The coding region of Mad1^597–719 was cloned by USER^® (NEB) into a modified pRSFDuet‐1 vector (71341‐3, Sigma‐Aldrich) with an N‐terminal double Strep His₆‐tag, followed by a tobacco etch virus (TEV) protease cleavage site (Demple & Linn, 1982 (link); Bitinaite et al,1992 ). Mad1 mutants were generated using the QuikChange™ Lightning Site‐Directed Mutagenesis Kit (Agilent), developed by Stratagene Inc. (La Jolla, CA) (Nøhr & Kristiansen, 2003 (link)). Mad1 constructs were transformed into Rosetta™ 2 (DE3) Singles™ Competent Cells (71400, Novagen) for expression. Expression was induced with 0.5 mM IPTG and grown overnight at 18°C. Cells were lysed in 25 mM HEPES pH 8.1, 250 mM NaCl, 2 mM DTT, 5% glycerol, 2 mM EDTA supplemented with lysozyme, and Complete™ EDTA‐free protease inhibitors (Roche). Proteins were purified over a Strep‐Tactin Superflow Plus column (QIAGEN) and cleaved with TEV protease overnight at 4°C. The cleaved Mad1^CTD was then diluted to 50 mM NaCl, purified over an anion exchange (Resource Q, GE Healthcare) column, followed by size exclusion chromatography using a Superdex 75 Increase column (GE Healthcare). Mad1^CTD was concentrated to 35 mg/ml in a buffer of 20 mM HEPES pH 7.5, 100 mM NaCl and 1 mM TCEP.

Fischer E.S., Yu C.W., Bellini D., McLaughlin S.H., Orr C.M., Wagner A., Freund S.M, & Barford D. (2021). Molecular mechanism of Mad1 kinetochore targeting by phosphorylated Bub1. EMBO Reports, 22(7), e52242.

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