For laboratory-scale expression of mAbs, plasmids bearing chimeric antibody heavy and light chain genes were prepared (EndoFree plasmid mega prep kit; Qiagen) and transfected into human embryonic kidney cells (HEK293-F) (Life Technologies) using polyethylenimine (PEI). Transfections were carried out using 1 mg of total DNA (500 μg each of VH and Vκ plasmid DNAs) and 1 L of the cultured HEK293-F cell suspension maintained in sterile Freestyle 293 expression medium (Invitrogen) without antibiotics at 37°C with 8% CO2, with shaking at 125 rpm. The transfected cells were grown for 8 days and purified using ProSep A beads (Millipore) and Econo-Pac chromatography columns (Bio-Rad). Recombinant mAbs were eluted in 100 mM glycine (pH 3.0) before neutralization with 1 M Tris-HCl (pH 8.0). Purified mAbs were quantified by SDS-PAGE and A280 measurements.
Endofree plasmid megaprep kit
Manufactured by Qiagen
Sourced in Germany
The EndoFree Plasmid Megaprep Kit is a laboratory equipment product designed for the isolation and purification of high-quality, endotoxin-free plasmid DNA from bacterial cultures. It utilizes a silica-based membrane technology to efficiently capture and purify the plasmid DNA, while effectively removing endotoxins and other contaminants.
Automatically generated - may contain errors
Lab products found in correlation
In fusion kit, by Takara Bio (2 mentions)
Hek293f, by Thermo Fisher Scientific (2 mentions)
Freestyle 293 expression medium, by Thermo Fisher Scientific (1 mentions)
Econo pac, by Bio-Rad (1 mentions)
Platinum super mix, by Thermo Fisher Scientific (1 mentions)
Akta purifier system, by GE Healthcare (1 mentions)
Nupage novex 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
One shot top10 chemically competent e coli, by Thermo Fisher Scientific (1 mentions)
Hek293e, by American Type Culture Collection (1 mentions)
6 protocols using endofree plasmid megaprep kit
1
Transient Expression of Monoclonal Antibodies in HEK293-F Cells
2
Construction of rAAV8 Vector Encoding Mouse Albumin
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A mouse genomic Alb segment (90474003-90476720 in NCBI reference sequence: NC_000071.6) was PCR-amplified and inserted between AAV2 ITRs into BsrGI and SpeI restriction sites in a modified pTRUF backbone31 (link). The genomic segment spans 1.3 Kb upstream and 1.4 Kb downstream to the Alb stop codon. We then inserted into the Bpu10I restriction site an optimized P2A coding sequence preceded by a linker coding sequence (glycine-serine-glycine) and followed by an NheI restriction site. Finally, we inserted a codon optimized hF9 cDNA into the NheI site to get pAB269 that served in the construction of the rAAV8 vector. To construct the inverse control, we first amplified an internal segment from the BsiWI restriction site to the 3′ NheI restriction site. PCR primers used had 15 base tails to allow subsequent integration of the amplicon into a BsiWI and NheI cleaved plasmid at the inverse orientation using an In-Fusion Kit (Clontech). Primers used were: For: 5′-ATGCAAGGCACGTACGTTATGTCAGCTTGGTCTTTTCTTTGATCC-3′ and Rev: 5′-TTTAGGCTAAGCTAGCTTTACTATGTCATTGCCTATGGCTATGAAGTG-3′. Final rAAV production plasmids were generated using an EndoFree Plasmid Megaprep Kit (Qiagen).
3
Construction of rAAV8 Vector Encoding Mouse Albumin
4
Recombinant Lundep Protein Production
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PCR fragments coding for Lundep (AY455916) were amplified (Platinum Supermix; Invitrogen) from Lu. longipalpis SG cDNA using gene-specific primers designed to amplify the mature peptide and added a 6x-His tag before the stop codon. PCR-amplified product was cloned into a VR2001-TOPO vector (modified version of the VR1020 vector; Vical Incorporated) and the sequence and orientation verified by DNA sequencing. Plasmid DNA (5 mg; VR2001-Lundep construct) was obtained using EndoFree plasmid MEGA prep kit (Qiagen, Valencia, CA) and filter sterilized through a 0.22-µm filter. Recombinant Lundep was produced by SAIC Advanced Research Facility (Frederick, MD) transfecting FreeStyle 293-F cells. Transfected cell cultures were harvested after 72 h and the supernatant shipped frozen to our laboratory until further processing. Recombinant protein expression was carried out by affinity and size-exclusion chromatography as described elsewhere [32] (link). Protein identity and purity was determined by Edman degradation and mass spectrometry.
5
Production and Purification of Recombinant LuloHya
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LuloHya coding DNA sequence (AF132515) was codon-optimized for mammalian expression and synthesized by BioBasic Inc. VR2001-TOPO vector containing LuloHya sequence (Vical Incorporated) was transformed in One Shot TOP10 Chemically Competent E. coli (Invitrogen). Plasmid DNA was prepared using the EndoFree plasmid MEGA prep kit (Qiagen, Valencia, CA). Recombinant protein expression was carried out at the SAIC Advance Research Facility (Frederick, MD). Briefly, human embryonic kidney cells (HEK293E; American Type Culture Collection, Manassas, Virginia) were transfected with 1 mg of plasmid DNA and supernatants were collected 72 h after transfection and shipped frozen to our laboratory for protein purification. Recombinant LuloHya was purified by affinity chromatography followed by size-exclusion chromatography, using Nickel-charged HiTrap Chelating HP and Superdex 200 10/300 GL columns, respectively (GE Healthcare Life Science, Piscataway, NJ). All protein purification experiments were carried out using the AKTA purifier system (GE Healthcare Life Science, Piscataway, NJ). Purified protein was separated in a NuPAGE Novex 4–12% Bis-Tris Protein Gels (Life Technologies) and visualized by Coomassie stain. Protein identity was verified by Edman degradation at the Research Technologies Branch, NIAID, NIH.
6
Production and Characterization of AAV8 Vectors
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All AAV8 vectors were produced by triple plasmid transfection of human embryonic kidney 293 cells (ATCC). The genome titer (genome copies mL–1) of AAV8 vectors was carried out by qPCR (Roche, Cat# 06402682001). The presence of endotoxins in AAV8 vector production was detected with LAL Chromogenic Endotoxin Quantitation Kit (Xiamen Bioendo Technology, China, Cat# EC32545S) according to the manufacturer’s manual. All recombinant AAV production plasmids were generated by using an EndoFree Plasmid Megaprep Kit (QIAGEN, Hilden, Germany). The sequences of primers were listed in Table S1 .
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