Codon optimized HTLV-1 Gag sequence, subcloned into the pCMV expression vector was ordered from Thermo Fisher Scientific. 293T cells, cultured in 10 × 10cm dishes, were transfected with the expression plasmid using Lipofectamine LTX Reagent with PLUS Reagent (Thermo Fisher Scientific, no. 15338100) according to manufacturer’s instructions. Cultivation medium, containing released HTLV-1 Gag-based VLPs was harvested 40 hours post transfection by ultracentrifugation. This and all further centrifugation steps were performed at 4°C. Cultivation medium was first clarified by centrifugation for 5 min at 1500 × g and subsequent filtering through syringe mounted PVDF filter with 0.45 μm pore size (Merck Millipore). VLPs were pelleted through a 20% sucrose cushion at 125,000 × g for 120 min. The pellet was briefly air-dried and resuspended in 150 μl PBS. Pooled and resuspended pellets were applied on a 6–18% Optiprep gradient, and centrifuged at 235,000 g for 90 min. Fractions containing Gag-based VLPs were pooled, diluted 1:4 in PBS, and pelleted without a cushion at 260,000 × g, for 45 min. The final pellet was then resuspended in 12 μl of PBS.
Pcmv expression vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PCMV expression vector is a DNA plasmid designed for the expression of recombinant proteins in mammalian cell lines. The vector features a cytomegalovirus (CMV) promoter, which drives high-level expression of the gene of interest. The vector also contains an antibiotic resistance gene for selection of transfected cells.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using pcmv expression vector
1
Purification of HTLV-1 Gag VLPs
2
Recombinant Decorin Production in CHO Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For proteoglycan production a pCMV expression vector (Thermo Fisher Scientific, Waltham, MA, USA) construct containing the human decorin cDNA sequence was created (Szilák Laboratories, Bioinformatics & Molecule-design Ltd. Szeged, Hungary). Next, CHO-S (Chinese hamster ovary) cells (Gibco/Thermo Fisher) were transfected by the expression vector using electroporation by Neon™ transfection system, following manufacturer's recommendations (Invitrogen/Thermo Fisher). Successfully transfected cells were selected by adding 500 μg/ml of G418 solution to cell culture medium (Sigma) and human recombinant decorin production was conducted following manufacturer's recommendations (Gibco/Thermo Fisher). The recombinant proteoglycan was isolated as previously described [28] (link). After chromatographic purification the samples were dialyzed against distilled water and the proteoglycan content was checked by dimethyl methylene blue (DMB, Sigma-Aldrich, St. Luis, MO, USA) staining. Finally, salts were added to the samples to convert the distilled water to 1x phosphate buffered saline (PBS), and the final decorin concentration was adjusted to 1000 μg/ml. These stock solutions were stored at -20˚C until use.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!