Centricon concentrator

Manufactured by Merck Group

Sourced in United States

Centricon concentrators are a type of lab equipment used for the concentration and purification of biological samples. These devices utilize centrifugal force to separate and concentrate macromolecules, such as proteins or nucleic acids, from a solution.

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Centricon (115 citations) Centricon-10 concentrator (8 citations) Amicon centricon concentrators (7 citations) Centricon Plus 70 concentrator (4 citations) Centricon spin concentrator (3 citations) Centricon YM-50 concentrator (2 citations)

10 protocols using centricon concentrator

Reconstitution of Iron-Sulfur Cluster in IssA

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IssA reconstitution was carried out by adding ferrous ammonium sulfate (10 mM) and sodium sulfide (10 mM) in buffer A to apo-IssA (0.25 mM) and incubating with shaking for 1 h at 80 °C. Excess iron and sulfide were removed by buffer exchange using a Centricon concentrator (Millipore) with a 10 kDa cut-off.

Vaccaro B.J., Clarkson S.M., Holden J.F., Lee D.W., Wu C.H., Poole II F.L., Cotelesage J.J., Hackett M.J., Mohebbi S., Sun J., Li H., Johnson M.K., George G.N, & Adams M.W. (2017). Biological iron-sulfur storage in a thioferrate-protein nanoparticle. Nature Communications, 8, 16110.

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Rac1 Cysteine Thiol Modification Kinetics

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Rac1^WT and Rac1^C18S were dialyzed into reducing buffer (15 mM HEPES (pH 8.0), 50 mM NaCl, 5 mM MgCl₂, 10 μM GDP, and 10 mM DTT), and the protein was incubated under reducing conditions for 30 min. Rac1 was then buffer exchanged using a Centricon concentrator (15-kDa molecular weight cut-off, Millipore) into nonreducing buffer (15 mM MES (pH 6.5), 30 mM NaCl, 5 mM MgCl₂, 200 μM DTPA, and 10 μM GDP). Rac1 was diluted into a mixed buffer system with pH values ranging from 5.5 to 8.5; each buffer contained 30 mM MES, 30 mM MOPS, 30 mM Tricine, 5 mM MgCl₂, 50 mM KCl, and 200 μM DTPA. ABD-f (4-(aminosul-fonyl)-7-fluoro-2,1,3-benzoxadiazole; Anaspec; Fremont, CA, USA) was added (1 mM) to initiate cysteine thiol modification. The reaction rate was determined by monitoring ABD-f fluorescence (excitation 389 nm, emission 513 nm) using a Spectromax M5e spectrometer (Molecular Devices; Sunnyvale, CA, USA). The initial reaction rates were fitted using linear regression analysis (Prism 5.0; GraphPad).

Hobbs G.A., Mitchell L.E., Arrington M.E., Gunawardena H.P., DeCristo M.J., Loeser R.F., Chen X., Cox A.D, & Campbell S.L. (2014). Redox regulation of Rac1 by thiol oxidation. Free radical biology & medicine, 79, 237-250.

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Purification and Biotinylation of apoA-II Deficient HDL

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The affinity of recombinant C57 and FVB apoA-II proteins for HDL was also measured using Surface Plasmon Resonance (SPR) on a BIAcore 3000 biosensor. HDL was purified from apoA-II deficient mice on the C57 background (Jackson Labs, Bar Harbor, ME). Mouse plasma was isolated by density gradient centrifugation. Plasma was brought to a density of 1.16 using NaBr to a final volume of 4 ml on top of which was layered 4 ml each of 1.125 and 1.063 NaBr density solutions. Density gradients were centrifuged for 17 hrs at 178,000× g and fractionated into 16 equal fractions. HDL fractions (by enzymatic cholesterol analysis, Roche Diagnostics, Indianapolis, IN) were combined and brought to 6 ml 1.25 density with NaBr and layered under 1.21 density solution and re-centrifuged. Purified apoA-II deficient HDL was then dialyzed/concentrated into PBS using a 100 kd cut-off Centricon concentrator (Millipore, Billerica, MA) and stored at 4°C. HDL protein concentration was determined by BioRad protein assay (Hercules, CA). Apoa2^−/− HDL was biotinylated by incubating 0.5 mg HDL with a 100 fold molar excess of EZ-Link NHS-PEG4-Biotin (Pierce, Rockford, IL) for 4 hours on ice followed by dialysis into PBS. HDL biotinylation was 50% by avidin-agarose pull down (Pierce, Rockford, IL).

Sontag T.J, & Reardon C.A. (2014). Polymorphisms of Mouse Apolipoprotein A-II Alter Its Physical and Functional Nature. PLoS ONE, 9(2), e88705.

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Bax (α2–α5) Incorporation into Bicelles

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A 6.78 mg of 1,2‐Dimyristoyl‐sn‐Glycero‐3‐Phosphocholine (DMPC) (Avanti Polar Lipids) was mixed with 51 μl of 200 mg/ml 1,2‐Dihexanoyl‐sn‐Glycero‐3‐Phosphocholine (DHPC) (Avanti Polar Lipids) in water to reach a ˜ 1:2 DMPC:DHPC molar ratio. The lipid mixture was subjected to cycles of freeze–thaw, pipetting and vortex until the lipids were fully dissolved, and the bicelles were formed homogeneously. The purified Bax (α2–α5) (0.1 mM) was incubated with the bicelles under 20‐rpm rotation at 25°C for 30 min. Five to eight aliquots of 0.1 mM Bax (α2–α5) in bicelles were mixed and concentrated to ˜ 500 μl using Centricon concentrator (EMD Millipore; MWCO, 3.0 kDa). Accordingly, the final sample for NMR measurements contains 0.5‐ 0.8 mM (monomer concentration) Bax (α2–α5), 50–80 mM DMPC, 100–160 mM DHPC, 100 mM NaCl, 10 mM MES pH 6.0, 0.1% NaN₃, and 10% D₂O. The DMPC: DHPC ratio was monitored by 1D NMR and the molar ratio (q) was kept 0.5–0.6 for all the NMR samples. DHPC and DMPC with deuterated acyl chains were used to prepare the samples for protein–protein NOESY experiments.

Lv F., Qi F., Zhang Z., Wen M., Kale J., Piai A., Du L., Wang S., Zhou L., Yang Y., Wu B., Liu Z., del Rosario J., Pogmore J., Chou J.J., Andrews D.W., Lin J, & OuYang B. (2021). An amphipathic Bax core dimer forms part of the apoptotic pore wall in the mitochondrial␣membrane. The EMBO Journal, 40(14), e106438.

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Bax (α2-α5) Incorporation into Bicelles

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) in Bicelles 6.78 mg of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) (Avanti Polar Lipids) were mixed with 51 μL of 200 mg/mL 1,2-Dihexanoyl-sn-Glycero-3-Phosphocholine (DHPC) (Avanti Polar Lipids) in water to reach a ~1:2 DMPC:DHPC molar ratio. The mixture was subjected to cycles of freeze-thaw, pipetting and vortex until the lipids were fully dissolved and the bicelles were formed homogeneously. The purified Bax (α2-α5) (0.1 mM) was incubated with the bicelles under 20-rpm rotation at 25 °C for 30 min. Five to eight aliquots of 0.1 mM Bax (α2-α5) in bicelles was mixed and concentrated to ~500 μL using Centricon concentrator (EMD Millipore; MWCO, 3.0 kDa). Accordingly, the final sample for NMR measurements contains 0.5-0.8 mM (monomer concentration) Bax (α2-α5), 50-80 mM DMPC, 100-160 mM DHPC, 100 mM NaCl, 10 mM MES pH 6.0, 0.1% NaN3, and 10% D2O. The DMPC: DHPC ratio was monitored by 1D NMR and the molar ratio (q) was kept 0.5-0.6 for all the NMR samples. DHPC and DMPC with deuterated acyl chains were used to prepare the samples for proteinprotein NOESY experiments.

Lv F., Qi F., Zhi Z., Wen M., Piai A., Du L., Zhou L., Yang Y., Wu B., Liu Y., Rosario J., Chou J.J., Andrews D.W., Lin J., & OuYang B. (2020). Amphipathic Bax core dimer forms part of apoptotic pore wall in the mitochondrial membrane.

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Purification of Recombinant GST-Synuclein

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The GST-synuclein fusion constructs were transformed into BL21 (DE3) pLysS strain of Escherichia coli and protein expression was induced with 1 mM IPTG. The recombinant GST-synuclein fusion proteins of WT and 112-syn were purified by affinity chromatography using glutathione sepharose-4B beads as described previously [38] . The fusion proteins were subjected to thrombin cleavage and subsequently mixed with glutathione sepharose-4B beads and p-aminobenzamidine agarose to trap any cleaved-off GST and thrombin. The proteins were concentrated using Centricon concentrators (Millipore, USA) of molecular mass cut-off 10 kDa and further purified using Superdex75 (GE Healthcare) gel-filtration column equilibrated with phosphate-buffered saline, pH 7.4. The protein concentrations were determined by bicinchoninic acid (BCA) protein assay kit [Thermo Fisher Scientific, IL, USA] using bovine serum albumin as a standard and the purity of the proteins was verified by SDS-PAGE. Protein samples were centrifuged at 12,000×g, for 30 min at 4°C prior to the experiments to remove any possible particulates.

Manda K.M., Yedlapudi D., Korukonda S., Bojja S, & Kalivendi S.V. (2014). The Chaperone-Like Activity of α-Synuclein Attenuates Aggregation of Its Alternatively Spliced Isoform, 112-Synuclein In Vitro: Plausible Cross-Talk between Isoforms in Protein Aggregation. PLoS ONE, 9(6), e98657.

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Purification and Characterization of Enzymes

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Aprotinin, pepstatin, leupeptin, phenylmethylsulfonyl fluoride (PMSF), tricine, ampicillin, DEAE-Sephacel, Ultrogel Aca-44, β-mercaptoethanol, pyridoxal 5’-phosphate, succinyl-CoA, α-ketoglutarate dehydrogenase, HEPES-free acid, MOPS, thiamine pyrophosphate and NAD⁺ were obtained from Sigma-Aldrich Chemical Company. Glucose, glycerol, acetic acid, methanol, glycine, disodium ethylenediamine tetraacetic acid dihydrate, tricine, ammonium sulfate and potassium hydroxide were purchased from Fisher Scientific. Centricon concentrators were from Millipore. SDS-PAGE reagents and Phusion DNA Polymerase were acquired from Thermo Scientific. 5-Aminolevulinic acid (ALA) hydrochloride was purchased from Acros Organics. Bmtl, Sall, BlpI, and BamHI restriction enzymes were obtained from New England BioLabs, Inc. Oligonucleotides were synthesized by Integrated DNA Technologies. T4 DNA ligase and ligase buffer were obtained from Thermo Scientific Fermentas and bicinchoninic acid protein determination kits were purchased from Thermo Scientific Pierce.

Fratz E.J., Clayton J., Hunter G.A., Ducamp S., Breydo L., Uversky V.N., Deybach J.C., Gouya L., Puy H, & Ferreira G.C. (2015). Human Erythroid 5-Aminolevulinate Synthase Mutations Associated with X-Linked Protoporphyria Disrupt Conformational Equilibrium and Enhance Product Release. Biochemistry, 54(36), 5617-5631.

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Generation of MHC Tetramers for T Cell Analyses

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The gp70_423–431 peptide (also known as AH1, SPSYVYHQF)-loaded H-2L^d MHC tetramers were obtained from the NIH Tetramer Core Facility. Tetramers were titrated to ensure an excess of tetramer was added to each sample when staining. Biotinylated H-2L^d monomers loaded with the gp70_423–431 peptide were also obtained from the NIH Tetramer Core Facility and tetramerized with a PE and oligonucleotide-barcode-conjugated streptavidin molecule (BioLegend, TotalSeq-A0951 PE Streptavidin product # 405251). Barcoded MHC tetramers were purified after tetramerization using fast protein liquid chromatography on an AKTA Pure Purification System equipped with a Superdex 200 column to isolate tetramerized MHC molecules from monomers and other smaller products. Using Unicorn version 6.3 software, the samples were run over the column at 0.75 mL/minute in 1X PBS (Life Technologies) with 0.1% sodium azide (Sigma) and tetramer-containing fractions were collected and pooled. Fractions were concentrated with Centricon concentrators (Millipore) to approximately 1 mg/mL.

Hay Z.L., Knapp J.R., Magallon R.E., O'Connor B.P, & Slansky J.E. (2023). Low TCR Binding Strength Results in Increased Progenitor-like CD8+ Tumor-Infiltrating Lymphocytes. Cancer Immunology Research, 11(5), 570-582.

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Characterization of Human Serum Albumin

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5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), DL-homocysteine thiolactone (HTL) hydrochloride, and all solvents and other reagents, unless stated differently, were purchased from Sigma (St. Louis, MO, USA) at the highest available grade and used without purification. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay kit was purchased from Invitrogen (Waltham, MA, USA). Centricon concentrators with a 3 kDa molecular weight cut-off were purchased from Millipore. Perfluoro-m-xylene (PFX) was prepared and kindly provided by V.E. Platonov (Novosibirsk Institute of Organic Chemistry, SB RAS). Cyanine dye derivatives were obtained from Lumiprobe (Moscow, Russia). Sequencing-grade modified trypsin for MALDI ToF peptide analysis was purchased from Promega (Madison, WI, USA) (cat. no. V5111). Trypsin for protein trypsinolysis was obtained from Gibco (Carlsbad, CA, USA) (cat. no. 15090046). The human serum albumin (HSA) used in this study was purchased from Sigma–Aldrich (St. Louis, MO, USA) (cat. no. A3782). MS (MALDI ToF) m/z HSA 66.48 kDa. Ellman’s test (SH group content) indicated that HSA in this study contained 0.28 ± 0.05 sulfhydryl groups per protein molecule.

Mitin D.E, & Chubarov A.S. (2023). Fluorinated Human Serum Albumin as Potential 19F Magnetic Resonance Imaging Probe. Molecules, 28(4), 1695.

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Purification of Circularized Nanowires

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A 50 mL reaction was prepared with 10 µM NWs and 5 µM (final concentrations) freshly made evolved sortase (Addgene plasmid # 75144, a gift from David Liu) in 300 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 10 mM CaCl₂. The reaction was incubated at 37°C for 3–4 hours or at 4°C overnight with gentle shaking on a rotating platform. A covalent sortase inhibitor AAEK2²⁰ was added to a concentration of 500 µM, and the solution was incubated for another 30 minutes at room temperature with gentle shaking. Proteins that did not undergo circularization were removed by binding to a Ni²⁺-NTA column. Circularized NWs (cNWs) were further purified by size-exclusion chromatography (Superdex 75 16/60) equilibrated in buffer containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl containing 50 mM sodium cholate or 1 mM dodecyl-β-D-maltoside (DDM). Purified protein was exchanged into buffer A_ix (20 mM Tris, pH 8.2, 1 mM DDM) using centricon concentrators (10 kDa MW cutoff, Millipore) and was then applied to a Resource Q column equilibrated with the same buffer (buffer A_ix). A linear salt gradient from 0–60 % buffer B_ix (20 mM Tris, pH 8.2, 1 mM DDM, 1M NaCl) was applied. Circularized proteins were eluted around 150–200 mM NaCl.

Nasr M.L., Baptista D., Strauss M., Sun Z.Y., Grigoriu S., Huser S., Plückthun A., Hagn F., Walz T., Hogle J.M, & Wagner G. (2016). Covalently circularized nanodiscs for studying membrane proteins and viral entry. Nature methods, 14(1), 49-52.

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