DENV1 strain 8700828, DENV2 strain 454009A, DENV3 strain 8700829, and DENV4 strain 59201818 were maintained in C6/36 cells. In brief, monolayers of C6/36 cells were incubated with DENV at a multiplicity of infection (MOI) of 1 and then incubated in 5% CO2 at 28°C for 4 to 7 days. The culture medium was harvested, and cell debris was removed by centrifugation at 1200 rpm for 10 min. To increase the viral titer, the virus supernatant was collected and concentrated by centrifugation at 3000 rpm for 20 min at 4°C using Amicon Ultra Centrifugal Filters (100K) (Millipore). Virus stocks were stored below −70°C until use. Virus titer was determined by plaque forming assay using BHK-21 cells.
100k amicon ultra centrifugal filters
Manufactured by Merck Group
The Amicon® Ultra Centrifugal Filters are a type of laboratory equipment designed for sample concentration and buffer exchange. They utilize a centrifugal force to pass a sample through a semi-permeable membrane, allowing for the selective retention of molecules above a certain molecular weight threshold.
Automatically generated - may contain errors
Lab products found in correlation
Optipro sfm, by Thermo Fisher Scientific (2 mentions)
Pfastbac dual vector, by Thermo Fisher Scientific (2 mentions)
0.2 μm low protein binding syringe filters, by Pall Corporation (2 mentions)
Fugene 6, by Promega (2 mentions)
Centrifuge 5415r, by Eppendorf (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
7 protocols using 100k amicon ultra centrifugal filters
1
Preparation and Titration of DENV Stocks
2
Protein A Immunoprecipitation and Purification
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PK-rS1-Ig cells were grown at 5 × 105 cells/well in a 6-well tissue culture plate in serum-free medium (OptiPRO SFM; Invitrogen). At 72 h post-seeding, the protein-containing culture supernatants were harvested and soluble proteins were immunoprecipitated with protein A Sepharose CL-4B beads in the presence of protease inhibitors at 4 °C for 16 h. The beads were collected and washed as described above. The samples were subsequently eluted with 50 mM sodium citrate/50 mM glycine (pH 2.0) and neutralized with 1 M Tris-HCl (pH 8.0). The purified proteins were concentrated with Amicon Ultra centrifugal filters 100K (Millipore). Protein concentration was measured using a BCA protein assay (Pierce, Rockford, IL) and the final products were analyzed by western blotting to confirm target protein purification.
3
Immunoprecipitation of Secreted Proteins
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PK-rS1-cFc cells were grown in 100-mm-diameter tissue culture dishes to 90 % confluency in serum-free medium (OptiPRO SFM; Invitrogen). At 72 h post-seeding, the protein-containing cell culture supernatants were harvested, and soluble proteins were immunoprecipitated with Chicken IgY Precipitating Resin (GenScript, Piscataway, NJ, USA) according to the manufacturer’s protocol in the presence of protease inhibitors at 4 °C for 16 h. The beads were collected by centrifugation at 5,000×g (Eppendorf centrifuge 5415R, Hamburg, Germany) for 5 min at 4 °C and washed three times with 0.5 M NaCl in PBS. The samples were subsequently eluted with 50 mM sodium citrate/50 mM glycine (pH 2.0) and neutralized with 1 M Tris-HCl (pH 8.0). The purified proteins were concentrated with Amicon Ultracentrifugal filters 100K (Millipore). Protein concentration was measured using a Pierce BCA protein assay (Thermo Scientific, Rockford, IL, USA), and the final products were analyzed by western blotting to confirm target protein purification.
4
Recombinant PtIP-83Aa Protein Expression
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Example 5
The gene encoding PtIP-83Aa SEQ ID NO: 2 was subcloned into the pFastBac™ Dual vector (Invitrogen®) with the stop codon removed for C-terminal translation of a 10×-histidine tag addition (SEQ ID NO: 27) and the sequence of the histidine-tagged PtIP-83Aa polypeptide is set forth as SEQ ID NO: 2. This vector was transformed into DH10Bac cells to generate baculovirus. These baculovirus were used to infect sf9 insect cells and incubated for 72 hours at 27° C. The infected insect cells were harvested by centrifugation. The cell culture pellet was suspended with 100 mL of lysis buffer (1×PBS, 10% glycerol, with protease inhibitor and benzonase) and incubated at 4° C. for 5 min with stirring, then homogenizing twice. The lysate was centrifuged at 16000 rpm for 20 min. The supernatant was saved and loaded onto two 2 mL Ni-NTA Hi-Bind Resin (Novagen, cat #70666) columns pre-equilibrated with Elute buffer (1×PBS, 10% glycerol). The columns were then sequentially eluted with 10 mL of Elute buffer containing 10, 20, 50, and 250 mM of imidazole. Samples were analyzed by SDS-PAGE. The purified fractions (E250) were concentrated using 100K Amicon® Ultra Centrifugal Filters (Millipore) to ˜0.5 mg/mL and demonstrated insecticidal activity against CEW, ECB, FAW and SBL similar to the activity spectrum obtained using transient expressing leaf discs.
5
Lentiviral Particle Production and Purification
6
Purification of Insecticidal Protein PtIP-83Aa
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Example 5
The gene encoding PtIP-83Aa SEQ ID NO: 2 was subcloned into the pFastBac™ Dual vector (Invitrogen®) with the stop codon removed for C-terminal translation of a 10×-histidine tag addition (SEQ ID NO: 27) and the sequence of the histidine-tagged PtIP-83Aa polypeptide is set forth as SEQ ID NO: 2. This vector was transformed into DH10Bac cells to generate baculovirus. These baculovirus were used to infect sf9 insect cells and incubated for 72 hours at 27° C. The infected insect cells were harvested by centrifugation. The cell culture pellet was suspended with 100 mL of lysis buffer (1×PBS, 10% glycerol, with protease inhibitor and benzonase) and incubated at 4° C. for 5 min with stirring, then homogenizing twice. The lysate was centrifuged at 16000 rpm for 20 min. The supernatant was saved and loaded onto two 2 mL Ni-NTA Hi-Bind Resin (Novagen, cat #70666) columns pre-equilibrated with Elute buffer (1×PBS, 10% glycerol). The columns were then sequentially eluted with 10 mLof Elute buffer containing 10, 20, 50, and 250 mM of imidazole. Samples were analyzed by SDS-PAGE. The purified fractions (E250) were concentrated using 100K Amicon® Ultra Centrifugal Filters (Millipore) to ˜0.5 mg/mL and demonstrated insecticidal activity against CEW, ECB, FAW and SBL similar to the activity spectrum obtained using transient expressing leaf discs.
7
Lentiviral Particle Production and Purification
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Lentiviral particles were produced by cotransfecting 293T cells with pSIN-EF2-HLA-G1, pMD2.G, and psPAX2 plasmids using FuGENE 6 (Promega, Madison, WI). Vector-containing supernatants were collected 48 hours after transfection.The collected supernatants, for both retrovirus and lentivirus, were filtered with 0.2 μm low protein-binding syringe filters (Pall Corporation, Ann Arbor, MI), concentrated using 100K Amicon Ultra centrifugal filters (Millipore, Billerica, MA), and stored in aliquots at −80 °C until further use.
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