The culture medium was harvested for sucrose density gradient analysis 4 days after transfection of the full-length HCV RNA. Collected culture media were cleared by low-speed centrifugation, and passed through a 0.45-μm filter. Filtered culture media were concentrated 30-fold using an Amicon Ultra-15 device (Molecular cut-off: 1 × 105 Da; Millipore). Concentrated culture media with or without NP-40 pretreatment were layered onto a stepwise sucrose gradient (10–60 %, wt/vol) and centrifuged for 16 h in a SW50.1 rotor (Beckman) at 40,000 rpm with RNase A at 4 °C. After centrifugation, about 20 fractions were collected. The HCV RNA and core antigen levels in each fraction were determined by sandwich EIA and qRT-PCR, as described above.
Amicon ultra 15 device
Manufactured by Merck Group
Sourced in United States, Germany, Ireland
The Amicon®Ultra-15 device is a centrifugal filter unit designed for the concentration and desalting of protein and DNA samples. The device utilizes a regenerated cellulose membrane to selectively retain molecules above a specific molecular weight cutoff. This allows for the concentration of target analytes while removing smaller components, such as salts and buffer additives, through the filter.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Sw50.1 rotor, by Beckman Coulter (1 mentions)
E coli bl21 de3 strain, by Thermo Fisher Scientific (1 mentions)
Hiprep 16 60 sephacryl s 200 hr, by GE Healthcare (1 mentions)
Mercaptoethanol, by Merck Group (1 mentions)
Bio tek, by Agilent Technologies (1 mentions)
Tris hcl, by Merck Group (1 mentions)
Econo pac chromatography column, by Bio-Rad (1 mentions)
Sepharose cl 2b, by Bio-Rad (1 mentions)
Sepharose cl 2b, by Merck Group (1 mentions)
Hispur cobalt resin, by Thermo Fisher Scientific (1 mentions)
6 well plate, by Corning (1 mentions)
Digital science image station 440cf, by Kodak (1 mentions)
Limulus amoebocyte lysate assay, by Merck Group (1 mentions)
Histrap, by GE Healthcare (1 mentions)
Quick start bradford protein assay, by Bio-Rad (1 mentions)
10 protocols using amicon ultra 15 device
1
HCV RNA Density Gradient Purification
2
Urine Protein Concentration and Desalting
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Approximately, 50 ml of second-morning urine samples were collected using consistent procedures for sample collection, storage, and transportation (www.molmeth.org/protocols/MF43XA ). The samples were centrifuged, and supernatant was stored at −80°C until further processing. Desalting and concentration of urinary proteins were done using 3 kDa molecular weight cut-off spin column (Amicon ultra-15 device, Millipore, Billerica, macroalbuminuria) by centrifugation at 4000 × g at 4°C. The protein content was estimated by the Bradford method.
3
Purification of Recombinant Proteins from E. coli
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The E. coli BL21(DE3) was used to express recombinant proteins. Bacteria were grown at 37 °C till its OD600 reached 0.8 in LB media containing 100 μg/ml kanamycin. The expression of recombinant proteins was induced by adding 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) for 10 h at 28 °C. Bacteria were harvested and re-suspended in buffer A (0.05 mol/L NaAc, 0.1 mol/L NaCl, pH 5.8). After sonication and centrifugation at 13,000 rpm for 15 min at 4 °C, the supernatant was collected and filtered with 0.45 μm membranes. The crude protein sample was subjected to anion exchange resin Q column (GE Healthcare, Uppsala, Sweden), and then eluted with buffer B (0.05 mol/L NaAc, 0.4 mol/L NaCl, pH 5.8). The fractions containing target proteins were collected, dialyzed and loaded onto a cation exchange resin SP column (GE Healthcare, Uppsala, Sweden), and eluted with buffer containing 0.5 mol/L NaCl (0.05 mol/L sodium citrate, pH 3.0). Collected proteins were separated by 15% (wt/vol) SDS/PAGE followed by Coomassie blue staining, and the purity of each fraction was evaluated. The purified proteins were then pooled and concentrated using an Amicon Ultra-15 device (Millipore) with a 3-KDa membrane cutoff and spun at 3,000 × g for 30 min. Protein concentrations were then determined by the BCA method following the manufacturer’s protocol.
4
Purification and Characterization of Gp41-MinTT Protein from E. coli
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Gp41-MinTT was produced in E. coli BL21 DE3 strain (Invitrogen). Cells were transformed with the plasmid pET-21-d+ Min-TT and cultured in LB medium supplemented with ampicillin. Cells were harvested by centrifugation and lysed with 50 mM Tris-HCl, 100 mM NaCl, (pH = 8) buffer supplemented with 2 mg/mL of lysozyme. Crude extract was then treated with 0.5% of Triton X-100 and inclusion bodies were collected after centrifugation. Inclusion bodies were solubilized using an urea-containing buffer (8 M urea, 20 mM Tris-HCl, 500 mM NaCl, 30 mM imidazole, pH = 8). Gp41-MinTT protein was purified by sepharose-Ni2+ affinity chromatography (GE Healthcare). An additional gel filtration purification step (Hiprep 16/60 Sephacryl S200 HR, GE Healthcare) was performed in the presence of 1% sodium dodecyl sulfate, 20 mM Tris-HCl buffer (pH = 7). Purified gp41-MinTT protein was dialyzed against PBS and concentrated with a 3 kDa Amicon Ultra-15 device (Milipore) to reduce SDS concentration to <0.1%. Purity was assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and G-250 coomassie staining (Biorad).
5
Purification of Malic Enzymes ME1 and ME2
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The human ME2 protein was expressed in Escherichia coli BL21 stain using the PRH281 vector under the control of the trp promoter, which was induced with indol-3-acetic acid (IAA). ME2 was purified using ATP agarose affinity chromatography (Sigma, St Louis, MO, USA). The human ME1 protein was expressed in Escherichia coli BL21(DE3) using the pET21b vector under the control of the T7 promoter, which was induced with isopropyl -D-1-thiogalactopyranoside (IPTG). ME1 was purified using Ni-NTA agarose affinity chromatography (Sigma, St Louis, MO, USA). Using a 30 KDa cutoff Amicon®Ultra-15 device, the purified malic enzymes were dialyzed and concentrated against a storage buffer containing 30 mM Tris-HCl (pH 7.4) and 2 mM -mercaptoethanol (Merck Millipore, Billerica, MA, USA). The purity of the protein was determined using SDS-PAGE, and the concentration of the protein was determined using a commercial protein assay buffer (Bio-Rad lab, Inc., Hercules, CA, USA) based on the Bradford method, and the absorbance at 595 nm was detected using a multi-mode microplate reader Biotek® (Agilent, Santa Clara, CA, USA).
6
Size Exclusion Chromatography of Extracellular Vesicles
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We concentrated 60 ml of conditioned media by using an Amicon Ultra‐15 device (Merck‐Millipore) with a 3 kDa cut‐off filter until the volume was reduced to 1 ml. A SEC column was prepared with Sepharose CL‐2B (Sigma‐Aldrich) as described previously (Böing et al., 2014 ). Briefly, 21 ml of a mixture of 75% Sepharose CL‐2B and 25% PBS containing 0.32% sodium citrate (pH 7.4) was poured into an Econo‐Pac chromatography column (Bio‐Rad). The column was washed with 60 ml of PBS‐citrate to eliminate any ethanol residual from the gel. The resultant column had a diameter of 1.5 cm and a height of 6.2 cm. The sample was loaded on the column and 30 fractions of 500 μl were collected. We measured the absorbance at 280 nm to determine the relative protein content of each fraction with a NanoDrop 2000c (Thermo Scientific). Fractions 5 to 16 were concentrated with an Amicon Ultra‐15 device with a 3 kDa cut‐off filter and stored at −80°C for further analysis (Figure 1 ).
7
Purification of scFv Antibody Fragments
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Flow-cytometry-positive
scFvs were cloned into expression vector POE-myc. Details of cloning,
bacterial transformation, and induction are available inSupporting Information . Soluble scFv from periplasmic
extract was purified using HisPur Cobalt Resin (Life Technology, Grand
Island, NY). The periplasmic extract (40 mL) was first incubated with
1 mL resin for an hour with rotation, and then the resin was gravity-packed
in a column (1 × 1 cm). The resin column was washed with equilibration
buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole,
PH 7.4) until the A280 of flow through
reached a baseline. The scFv was subsequently eluted with 50 mM sodium
phosphate, 300 mM sodium chloride, 150 mM imidazole, pH 7.4. A few
fractions (1 mL each) of eluate were collected to ensure all protein
had been eluted, then those fractions with protein were pooled and
concentrated using an Amicon ultra-15 device (EMD Millipore, Billerica,
MA). The purified scFv was analyzed by SDS-PAGE and the protein concentration
was determined using the BCA protein assay (Pierce, Rockford, IL).
scFvs were cloned into expression vector POE-myc. Details of cloning,
bacterial transformation, and induction are available in
extract was purified using HisPur Cobalt Resin (Life Technology, Grand
Island, NY). The periplasmic extract (40 mL) was first incubated with
1 mL resin for an hour with rotation, and then the resin was gravity-packed
in a column (1 × 1 cm). The resin column was washed with equilibration
buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole,
PH 7.4) until the A280 of flow through
reached a baseline. The scFv was subsequently eluted with 50 mM sodium
phosphate, 300 mM sodium chloride, 150 mM imidazole, pH 7.4. A few
fractions (1 mL each) of eluate were collected to ensure all protein
had been eluted, then those fractions with protein were pooled and
concentrated using an Amicon ultra-15 device (EMD Millipore, Billerica,
MA). The purified scFv was analyzed by SDS-PAGE and the protein concentration
was determined using the BCA protein assay (Pierce, Rockford, IL).
8
Conditioned Media Preparation for Cytokine Analysis
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Conditioned media (CM), for subsequent array analysis, were obtained from the HUVEC and DU145 cell lines. Briefly, 0.3 × 106 cells were seeded into 6-well plates (Corning) in complete medium (EBM™ for HUVEC and RPMI for DU145) at 37 °C under 5% CO2. When cells reached 80% of confluence, HUVECs were stimulated for 1 h with TNFα (10 ng/mL), then both cell lines were washed for 30 min with medium without serum and were starved for 24 hours (h) for HUVECs and 48 h for DU145 cells in 1 mL/well serum-free medium, supplemented or not with Fuco (20 µg/mL). Finally, CM were collected, pulling two wells per condition, and residual cells and debris were discarded by centrifugation.
DU145 and LNCaP cells were cultured as above in T75 or T175 flasks to 80–85% confluence, rinsed with 1× phosphate-buffered saline (PBS), then cultured in their respective medium without FBS. CM were harvested after 48 h and filtered through the Amicon® Ultra-15 device (50 kDa, Merck Millipore, Darmstadt, Germany) to remove cells and debris. All CMs collected were stored at −80 °C.
DU145 and LNCaP cells were cultured as above in T75 or T175 flasks to 80–85% confluence, rinsed with 1× phosphate-buffered saline (PBS), then cultured in their respective medium without FBS. CM were harvested after 48 h and filtered through the Amicon® Ultra-15 device (50 kDa, Merck Millipore, Darmstadt, Germany) to remove cells and debris. All CMs collected were stored at −80 °C.
9
Western Blot and ELISA Analysis of IFNβ
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Cells were sonicated in radioimmunoprecipitation assay buffer (RIPA) or Laemmli buffer, and proteins were analyzed by Western blot, as described [28 (link)]. β-Actin level was also determined as a control. Western blot images were acquired using Kodak Digital Science Image Station 440CF. Monoclonal F/ARFP antibodies were generated against recombinant JFH1 F protein (J.-H. James Ou from University of Southern California). IFNβ concentrations in the cell culture medium were determined by ELISA with or without concentrating the samples, using Human IFNβ ELISA kit from Interferon Source, Inc. and using IFNβ as standards. Individual data points for ELISA are presented in S1 Table . For concentrating the samples, an Amicon Ultra-15 device (EMD Millipore) was used. Culture medium was pipetted into the Amicon Ultra-15 device and then centrifuged for 30 min at 3,000g (4°C). For immunofluorescence staining, samples were fixed, permeabilized, and incubated with primary antibodies, followed by incubation with fluorophore-conjugated secondary antibodies, and imaged by confocal laser scanning microscopy, as described [28 (link)]. Images were quantified by ImageJ available at http://rsbweb.nih.gov/ij/ . Individual data points for ImageJ quantification are presented in S1 Table .
10
Recombinant HSP60 Protein Purification
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rHSP60 was expressed and purified as described by Fernandes et al [15 (link)]. Briefly, E. coli transformed with pET28a– HSP60 vector were grown in LB medium supplemented with kanamycin sulfate (50 μg/mL). After inducing with 0.5 mM isopropyl-β-d -thiogalactopyranoside for 6 hours, bacterial cells were lysed by sonication in lysing buffer (50 mM NaH2PO4, 300 mM NaCl, 5 mM 2-mercaptoethanol, and 0.5% Triton X-100, pH 8.0). The sample was centrifuged at 7,000 × g and the pellet washed 5 times with the lysing buffer. Pellet inclusion bodies was solubilized in a denaturing buffer (50 mM NaH2PO4, 300 mM NaCl, 30 mM imidazole, 7 M urea, 5 mM 2-mercaptoethanol, and 0.5% Tween 20, pH 8.0) for 1 hour. After centrifugation at 7,500 × g, the supernatant was clarified through 0.22 μm filter, and submitted to a Ni2+–Sepharose affinity column (His-Trap; GE Healthcare). rHSP60 purified was concentrated and refolded by dialysis against phosphate-buffered saline (PBS) in a Amicon Ultra 15 device with a molecular weight cut-off of 10-kDa (Merck, Cork, Ireland). Protein concentration was determined using Quick Start Bradford Protein Assay (Bio-Rad, Hercules, USA). Purified rHSP60 sample was analyzed by SDS-PAGE. The sample contained less than 0.05 ng/mL of bacterial endotoxin, as determined by the Limulus amoebocyte lysate assay (Sigma-Aldrich, St. Louis, USA).
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