Sephacryl s 200 column

Manufactured by GE Healthcare

Sourced in United States, United Kingdom, Sweden

The Sephacryl S-200 column is a size-exclusion chromatography medium designed for the separation and purification of proteins, enzymes, and other biomolecules. It is composed of cross-linked allyl dextran and N,N'-methylene bisacrylamide, providing a porous matrix for the separation of molecules based on their size and molecular weight.

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Lab products found in correlation

Resource s cation exchange column, by GE Healthcare (2 mentions) Zetasizer nano z, by Malvern Panalytical (1 mentions) Prolong gold, by Thermo Fisher Scientific (1 mentions) Kta prime system, by GE Healthcare (1 mentions) Infinite m200 pro plate reader, by Tecan (1 mentions) Amicon centricon concentrators, by Merck Group (1 mentions) E coli bl21 star de3 cells, by Thermo Fisher Scientific (1 mentions) Akta pure, by GE Healthcare (1 mentions) Sodium deoxycholate, by Avantor (1 mentions) Phenyl sepharose cl 4b, by GE Healthcare (1 mentions) Deae sephacel, by GE Healthcare (1 mentions) Fplc system, by GE Healthcare (1 mentions) Nickel affinity chromatography, by Qiagen (1 mentions) Hitrap q column, by GE Healthcare (1 mentions) Deae sepharose ff column, by GE Healthcare (1 mentions) Bme vitamins, by Merck Group (1 mentions)

18 protocols using sephacryl s 200 column

Measuring eEF1Bα Hydrodynamic Radius by DLS

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To measure a hydrodynamic radius of eEF1Bα we used the Dynamic Light Scattering technique (DLS) and the Adaptive Correlation approach described in (22 (link)). DLS experiments were performed using a Zetasizer Nano ZS (Malvern Panalytical Ltd, UK) at 25°C with a scattering angle of 173° in air. All samples (1 ml) were measured in a 1 cm glass cuvette. Briefly, three measurements for each eEF1Bα concentration were done. Each measurement included 30 sub-measurements with duration time of 1, 2 and 3 s. The steady-state sub-measurements were analyzed using cumulants analysis. The correlation functions of the steady-state sub-measurements were averaged to report the hydrodynamic radius (Z_ave) and polydispersity index (PdI) values. Z_ave values were obtained for four different eEF1Bα concentrations. R_H0 – a hydrodynamic radius of eEF1Bα at the infinite dilution was calculated from the plot of Z_ave versus eEF1Bα concentration by extrapolation to zero concentration.
Prior to the DLS measurements, eEF1Bα was subjected to gel filtration on a Sephacryl S200 column (GE Healthcare) equilibrated in buffer, containing 25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5% glycerol (v/v) and 1 mM dithiothreitol. Fractions with the highest eEF1Bα concentration were centrifuged at 16 000 g for 2.5 hours at 10°C. Buffer was prepared using ultrapure deionized water, filtered (pore size 0.2 μm) and degassed.

Bondarchuk T.V., Shalak V.F., Lozhko D.M., Fatalska A., Szczepanowski R.H., Liudkovska V., Tsuvariev O.Y., Dadlez M., El'skaya A.V, & Negrutskii B.S. (2022). Quaternary organization of the human eEF1B complex reveals unique multi-GEF domain assembly. Nucleic Acids Research, 50(16), 9490-9504.

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Mycobacterial Lipoglycan Purification and Characterization

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All chemicals were obtained from Sigma unless stated otherwise. For autophagy flux experiments, Torin 1 (Enzo life sciences, NY, USA) and chloroquine were used at 1 μM and 50 μM, respectively. The Protein A/G sepharose reagent used for immunoprecipitation experiments was from Rockland Immunochemicals (Rockland, NY, USA). Mannose-capped lipoarabinomannan was purified from Mycobacterium bovis BCG, as previously reported [75 (link),76 (link)]. Briefly, cells were harvested, washed in PBS, and disrupted by French press in 8% (v/v) Triton X-114, 5 mM EDTA, and 10 mM MgCl₂. After phase separation at 37 °C, lipoglycans were collected in the lower phase, precipitated by cold ethanol, and extracted by saturated phenol. ManLAM was then separated from other lipoglycans (lipomannan and phospho-inositol mannosides) by gel filtration on a Sephacryl S-200 column (GE Healthcare, Amersham, UK) irrigated with tris deoxycholate buffer (10 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.2 M NaCl, 0.25% deoxycholate). The purity of the eluted fractions was assessed by 13% SDS-PAGE and staining for carbohydrates. The structural integrity of the lipoglycan was checked by multi-nuclear magnetic resonance spectroscopy as previously described [76 (link)]. For immunofluorescence assays, DAPI (4′,6′ Di Amidino-2-Phényl Indole) and Prolong^®Gold were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

Papin L., Lehmann M., Lagisquet J., Maarifi G., Robert-Hebmann V., Mariller C., Guerardel Y., Espert L., Haucke V, & Blanchet F.P. (2023). The Autophagy Nucleation Factor ATG9 Forms Nanoclusters with the HIV-1 Receptor DC-SIGN and Regulates Early Antiviral Autophagy in Human Dendritic Cells. International Journal of Molecular Sciences, 24(10), 9008.

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Recombinant Protein Production and Purification

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pNgtS, pGH92 and pEndoD were transformed into the expression strain Escherichia coli BL21 Star (DE3). NgtS was expressed in 6 L yeast-tryptone broth overnight at 16°C with 0.5 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG). Recombinant SpGH92 was produced via the autoinduction method [60 (link)] by incubating inoculated 1 L cultures at 37°C for 96 hours. EndoD was expressed at 37°C for 3 hours with 0.5 mM IPTG induction as described by Muramatsu et al. [20 (link)]. For all proteins, cells were harvested by centrifugation at 5000 × g for 10 minutes and ruptured by chemical lysis. The cleared supernatant of the cell lysate was loaded onto a Ni²⁺-NTA immobilized metal affinity chromatography column. Polypeptide was eluted with binding buffer (20 mM Tris, 500 mM NaCl, pH 8.0) containing increasing concentrations of imidazole (0–500 mM). Protein was concentrated and buffer-exchanged into 20 mM Tris, pH 8.0 in an Amicon stirred ultrafiltration unit (EMD Millipore Co.) with a 10,000 Da molecular weight cut-off membrane. NgtS was further purified by size exclusion chromatography using a Sephacryl S-200 column (GE Healthcare, Little Chalfont, UK) and concentrated again using an Amicon. Protein concentration was determined by UV absorbance at 280 nm using the calculated extinction coefficients of 60280 M⁻¹ cm⁻¹ for NgtS, 121295 M⁻¹ cm⁻¹ for SpGH92 and 221065 M^-1 cm^-1 for EndoD.

Robb M., Hobbs J.K., Woodiga S.A., Shapiro-Ward S., Suits M.D., McGregor N., Brumer H., Yesilkaya H., King S.J, & Boraston A.B. (2017). Molecular Characterization of N-glycan Degradation and Transport in Streptococcus pneumoniae and Its Contribution to Virulence. PLoS Pathogens, 13(1), e1006090.

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Purification of Jatropha Trypsin Inhibitor

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The crude extract prepared as described in Section “protein Determination” was fractionated by precipitation with 2.5% (v/v) trichloroacetic acid (TCA) final concentration, at 4°C and centrifuged at 14,000 × g, 4°C, 30 min. The clear supernatant obtained was dialyzed exhaustively against water (Milli-Q grade), lyophilized, and assayed for antitrypsin activity. The TCA fraction (30 mg) was dissolved in 0.050 M sodium phosphate buffer/0.2 M NaCl, pH 7.5 and applied to a trypsin-Sepharose 4B column (11.5 cm × 2.2 cm) equilibrated with the above buffer. After complete removal of the non-retained proteins with the equilibrating buffer, the proteins bound to the immobilized trypsin were eluted with 0.1 M HCl, dialyzed exhaustively against water (Milli-Q grade) and lyophilized. This material (2 mg) was loaded on a Sephacryl S-200 column connected to an ÄKTA-Prime System (GE Healthcare) previously equilibrated and eluted with 0.050 M sodium phosphate buffer/0.2 M NaCl, pH 7.5. Fractions (1 mL) were eluted at the flow rate of 0.5 mL/min and the protein fractions obtained evaluated for trypsin inhibitory activity as described before. The purified trypsin inhibitor was named JcTI-I (J. curcas Trypsin Inhibitor I).

Costa H.P., Oliveira J.T., Sousa D.O., Morais J.K., Moreno F.B., Monteiro-Moreira A.C., Viegas R.A, & Vasconcelos I.M. (2014). JcTI-I: a novel trypsin inhibitor from Jatropha curcas seed cake with potential for bacterial infection treatment. Frontiers in Microbiology, 5, 5.

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Chaperone-Mediated GFP Fusion Protein Analysis

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Reaction mixtures (100 μL) containing GFP-15, GFP-X₃₀-H₆, or GFP-X₇-H₆ (0.4 μM) with or without ClpB_{E279A, E678A} or Hsp104_{E285A, E687A} (2 μM) were incubated in buffer A, 0.005% Triton-X100, 5 mM ATP, and 10 mM MgCl₂ for 45 min at room temperature. Reaction mixtures were fractionated on a Sephacryl S200 column (GE Healthcare) equilibrated with 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 20 mM KCl, 0.1 mM EDTA, 10% glycerol, 5 mM ATP, and 10 mM MgCl₂ at room temperature. Fractions (100 μL) were collected and GFP fluorescence was measured in a Tecan Infinite M200Pro plate reader at 25°C as described above. The percentage of the GFP fusion protein signal that was shifted upon chaperone binding was determined by calculating the area under the shifted peak compared to the total area under all peaks. The elution profile of ClpB_{E279A, E678A} or Hsp104_{E285A, E687A} (2 μM) was determined in the absence of GFP fusion protein by measuring protein in each fraction using the Bradford assay.

Johnston D.M., Miot M., Hoskins J.R., Wickner S, & Doyle S.M. (2017). Substrate Discrimination by ClpB and Hsp104. Frontiers in Molecular Biosciences, 4, 36.

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Reconstitution of LH2 with AcChl a and BChl a

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A solution of B800-depleted
LH2 in a mixed buffer of 20 mM Tris and 10 mM succinate containing
0.1% DDM (pH 8.0) was mixed with 1/100 volume of a methanol solution
of AcChl a or BChl a, followed by
incubation at 35 °C for 2 h in the dark. The sample was concentrated
by ultrafiltration using Amicon centricon concentrators (30 kDa cutoff,
Merk Millipore Ltd.) and was loaded onto a Sephacryl-S200 column (GE
Healthcare) in 20 mM Tris buffer containing 0.1% DDM and 150 mM NaCl
(pH 8.0). LH2 proteins collected were desalted by ultrafiltration
using Amicon centricon concentrators (30 kDa cutoff, Merk Millipore
Ltd.). LH2 proteins, which are reconstituted with AcChl a and BChl a, are hereafter denoted as AcChl-reconstituted
LH2 and BChl-reconstituted LH2, respectively.
The occupancy
of AcChl a in the B800 sites in AcChl-reconstituted
LH2 was estimated from electronic absorption spectra of extracted
chlorophyllous pigments in the Q_y region
in methanol, as reported elsewhere.^{19 (link)} The
occupancy of BChl a in the B800 sites in BChl-reconstituted
LH2 was estimated by comparing Q_y absorbance
of B800 BChl a in BChl-reconstituted LH2 with that
in native LH2.^{19 (link)}

Saga Y., Yamashita M., Imanishi M., Kimura Y., Masaoka Y., Hidaka T, & Nagasawa Y. (2020). Reconstitution of 3-Acetyl Chlorophyll a into Light-Harvesting Complex 2 from the Purple Photosynthetic Bacterium Phaeospirillum molischianum. ACS Omega, 5(12), 6817-6825.

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Recombinant Protein Expression in E. coli

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All recombinant expression vectors were transformed into E. coli BL21 Star (DE3) cells (Invitrogen). Fusion proteins were produced using LB medium supplemented with Ampicillin (50 µg/mL) for the StrH or with kanamycin (50 µg/mL) for the SP_2141 and SP_2146. Briefly, bacteria with the appropriate expression plasmid were grown at 37 °C until the OD₆₀₀ reached 0.6–0.8. Gene expression was then induced by adding isopropyl-b-D-1-thiogalacto pyranoside (IPTG) to a final concentration of 0.5 mM, and then incubated overnight at 16 °C with shaking. Cells were harvested by centrifugation and disrupted by Phenylmethanesulfonyl fluoride (PMSF) lysis buffer (1 × PBS, 1 mM PMSF). Proteins were purified by Ni2+ immobilized metal affinity chromatography, followed by size exclusion chromatography using a Sephacryl S-200 column (GE Health care, Chicago, IL, USA) and analyzed by 10% SDS-PAGE. Protein concentration was determined and stored at −80 °C in PBS.

Han X., Jin L., Zhao Z., Xu X., Liu S., Huang Y., Liu X., Xu Y., Yang D., Huang W, & Wang L. (2023). Combining the In Silico and In Vitro Assays to Identify Strobilanthes cusia Kuntze Bioactives against Penicillin-Resistant Streptococcus pneumoniae. Pharmaceuticals, 16(1), 105.

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Purification of NnNV Enzyme

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15 mg of NnNV dissolved in 10 mL of 20 mM of phosphate buffer (pH 7.4) containing 150 mM of NaCl was applied to a Sephacryl S-200 column (110 cm × 2.5 cm, GE Life Sciences, Chicago, IL, USA) connected to an FPLC system AKTA Pure (GE Life Sciences, Chicago, IL, USA). The column was equilibrated and eluted with 20 mM Tris–HCl (pH 8.0) containing 100 mM NaCl. Fractions of 3 mL/tube were collected, the flow rate was set to be 36 mL/h and the elution was monitored at 280 nm and 214 nm. Fractions were combined according to the absorbance at 280 nm. The combined fractions Peak A-I were concentrated by ultrafiltration (MWCO 3 kDa, Millipore, Burlington, MA, USA) and were subject to protein concentration determination, SDS-PAGE profiling and characterizing the enzymatic activities.

Yue Y., Yu H., Li R, & Li P. (2021). Topical Exposure to Nemopilema nomurai Venom Triggers Oedematogenic Effects: Enzymatic Contribution and Identification of Venom Metalloproteinase. Toxins, 13(1), 44.

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Recombinant Histone Purification Protocol

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Recombinant Xenopus laevis histone proteins were overexpressed in BL21 (DE3) pLysS Escherichia coli cells and purified from inclusion bodies with modifications to the established protocol [7] (link). Inclusion bodies for histones containing cysteine point mutants were solubilized in sulfitolysis buffer [20 mM Tris–HCl (pH 9.0), 6 M guanidine–HCl, 300 mM sodium sulfite, 60 mM sodium tetrathionate] for 24 h and 22 °C in the dark [27] (link). Denatured histones were purified by size-exclusion chromatography using a Sephacryl S-200 column (50 × 1000 mm; GE Healthcare) equilibrated in 20 mM sodium acetate (pH 5.2), 6 M guanidine–HCl, 200 mM NaCl, and 1 mM EDTA. Selected fractions were pooled, dialyzed extensively against water and lyophilized. Histones were redissolved in 2 M guanidine–HCl for purification by reversed-phase chromatography (TSK-Octadecyl-4PW, 21.5 × 150 mm; Tosoh Biosciences) and eluted with a 35%–65% gradient of buffer A (0.3% TFA, 5% acetonitrile) to buffer B (0.3% TFA, 90% acetonitrile). Selected fractions were pooled, dialyzed and lyophilized as 4-mg aliquots and stored at − 80 °C.

Frouws T.D., Barth P.D, & Richmond T.J. (2018). Site-Specific Disulfide Crosslinked Nucleosomes with Enhanced Stability. Journal of Molecular Biology, 430(1), 45-57.

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Purification of Lipase from C. rugosa

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Lipase type VII from C. rugosa was obtained from Sigma Chemicals Co. (St Louis, MO, USA), tributyrin and triacetin from Fluka (Deisenhofen, Germany), and sodium deoxycholate from Amresco (Solom, OH, USA). Gels for protein purification, DEAE-Sephacel, Phenyl-Sepharose CL-4B, Sephacryl HR 100, and the Sephacryl S200 column were from GE Healthcare (Piscataway, NJ, USA). All chromatographic steps were performed on a fast protein liquid chromatography (FPLC) system (Pharmacia Biotech, Sweden).

Álvarez-Cao M.E., González R., Pernas M.A, & Rúa M.L. (2018). Contribution of the Oligomeric State to the Thermostability of Isoenzyme 3 from Candida rugosa. Microorganisms, 6(4), 108.

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