293fectin transfection reagent

Manufactured by Thermo Fisher Scientific

Sourced in United States

293fectin is a transfection reagent designed for efficient delivery of nucleic acids, such as DNA and RNA, into mammalian cells. It facilitates the uptake of genetic material into the target cells, enabling researchers to study gene expression, protein production, and other cellular processes.

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293fectin (142 citations) Transfection reagent (92 citations) 293Fectin reagent (17 citations)

50 protocols using 293fectin transfection reagent

Recombinant Protein Expression and Purification

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Example 2

Protein Expression and Purification.

The recombinant proteins expressed in Escherichia coli (E. coli) BL21 (DE3) RIPL cells were purified according to previously described protocols^{10, 12}. Influenza HA4900 was purified from HEK293F suspension cells (Thermo Fisher Scientific®, MA) according to previous reports³¹. Briefly, the pAAV-ITRs-HA4900 plasmid was transiently transduced into HEK293F cells maintained in FreeStyle® 293 expression medium (Thermo Fisher Scientific®, MA) using the 293Fectin™ transfection reagent (Thermo Fisher Scientific®, MA). The cells were then incubated at 37° C. and 8% CO₂while shaking at 130 rpm overnight. After 12 h, an equal volume of fresh medium supplemented with sodium butyrate solution (enhancing protein expression, 2 nM final concentration) (Sigma-Aldrich®, MO) was added to the cells. On day 7, the pure supernatants were harvested by centrifugation and filtered over 0.22-μm filters (Sartorius Stedim Biotech, Germany). Proteins were purified using HisTrapHP and gel filtration columns (GE Healthcare™, IL).

US11155835B2. Prokaryotic-eukaryotic hybrid viral vector for delivery of large cargos of genes and proteins into human cells (2021-10-26). The Catholic University of America [US]. Inventors: Venigalla B. Rao [US], Jingen Zhu [US].

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Colorimetric Enzyme Assay Protocol

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2−Aminopyridine, copper sulfate, 1,5−diaminopentane dihydrochloride (cadaverine), dichloro(1,10−phenanthroline) Cu(II), horseradish peroxidase (HRP), 2−hydrazinopyridine (2HP) dihydrochloride, and phenylhydrazine (PH) hydrochloride and 4−(2−pyridilazo)resorcinol (PAR) were purchased from SigmaAldrich (St. Louis, MO, USA). FreeStyle™ 293−F Expression System (FreeStyle™ 293−F cells, FreeStyle™ 293 Expression Medium, 293 fectin™ Transfection Reagent, Opti−MEM™I Reduced Serum Medium) and N−acetyl−3,7−dihydroxyphenoxazine (Amplex™ Red Reagent) were obtained from ThermoFisher Scientific (Lenexa, KS, USA).

Meier A.A., Moon H.J., Sabuncu S., Singh P., Ronnebaum T.A., Ou S., Douglas J.T., Jackson T.A., Moënne-Loccoz P, & Mure M. (2022). Insight into the Spatial Arrangement of the Lysine Tyrosylquinone and Cu2+ in the Active Site of Lysyl Oxidase-like 2. International Journal of Molecular Sciences, 23(22), 13966.

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Production of SARS-CoV-2 Spike Trimer Protein

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An uncleaved version of the ectodomain (residues 1–1208) of SARS-CoV-2 spike protein, namely SARS-CoV-2 S-2P was constructed as sorting probe that contains the following modifications - D614G mutaiotn, ₆₈₂RRAR₆₈₅ →SGAG substitution at the furin cleavage site, and two proline substitutions ₉₈₆ KV₉₈₇→PP. Additionally in the S-2P construct, the C terminus of the S ectodomain is appended with a GSG peptide linker, a foldon trimerization motif, an HRV3C protease cleavage site, an 8 × His Tag, and a TwinStrep Tag (SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK) as previously described.^{75 (link),76 (link)} The S-2P protein encoding sequence were codon optimized for human cell expression (GenScript, NJ), cloned into the mammalian cell expression vector pcDNA3.1(−), and confirmed by sequencing prior to transient transfection in FreeStyle 293-F cells with 293fectin transfection reagent (Thermo Fisher Scientific). Culture supernatants were harvested at 5 days post transfection, filtered, and purified by StrepTactin resin (IBA Lifesciences). Elutes were subjected to size exclusion chromatography using a Superpose 6 10/300 GL column (Sigma-Aldrich, MO) to collect trimer fraction. Elutes from both purification procedures were concentrated and buffer exchanged with phosphate-buffered saline (PBS) with an Amicon Ultra 10 kDa molecular weight cutoff concentrator (Millipore).

Lopes de Assis F., Hoehn K.B., Zhang X., Kardava L., Smith C.D., El Merhebi O., Buckner C.M., Trihemasava K., Wang W., Seamon C.A., Chen V., Schaughency P., Cheung F., Martins A.J., Chiang C.I., Li Y., Tsang J.S., Chun T.W., Kleinstein S.H, & Moir S. (2023). Tracking B cell responses to the SARS-CoV-2 mRNA-1273 vaccine. Cell reports, 42(7), 112780.

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Lentiviral Transduction of HEK293TN Cells

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HEK293TN cells (System Biosciences, Mountain View, CA) were grown in DMEM high glucose medium (Hyclone, GE Healthcare Life Sciences, Pittsburgh, PA)) supplemented with 10% FBS, 2 mM L-Glutamine, 25mM Hepes, and 1× non-essential amino acids (NEAA, Thermo). 24 hours before transfection, 8 – 9 × 10⁶ cells were plated in a 150 mm tissue-culture dish. For transfection, 13.5 μg of the GIPZ lentiviral shRNA plasmid expressing a scrambled RNA control was combined with 8.8 μg of the packaging vector psPAX2 and 4.7 μg of the envelope expressing plasmid pCMV-VSV-G. The DNA mixture was diluted in Opti-MEM I medium (Thermo Fisher) and admixed with 293fectin transfection reagent (Thermo Fisher) at a ratio of 3 μl per 1 μg of DNA and thereafter dropwise added to HEK293TN cells. Viral supernatant was collected at 60 h after transfection, filtered through a 0.45 μm PVDF filter and concentrated through ultracentrifugation (Sorvall, Thermo Fisher; 23,000 rpm, 4 °C, 2 hours). The concentrated lentiviral particles were resuspended over night in HBSS with 25 mM HEPES and stored at −80 °C.

Reinisch A., Thomas D., Corces M.R., Zhang X., Gratzinger D., Hong W.J., Schallmoser K., Strunk D, & Majeti R. (2016). A Humanized Ossicle-niche Xenotransplantation Model Enables Improved Human Leukemic Engraftment. Nature medicine, 22(7), 812-821.

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Lentiviral Transduction of HEK293TN Cells

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Recombinant Expression and Purification of BChE Variants

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Genomic DNA from clones 14 and 15 was extracted as described previously (Lõoke et al., 2011 (link)) and used as a PCR template with primer pair a/b: (a) GCTTACTCGGAAGATGACATCATAATTGCAACAA (b) CTCCTTCGATCGACGCATGCAGAAAGCTCTGG. The product was cloned into the pFUSE-BChE-6xHis plasmid using NheI and NcoI restriction sites. These plasmids were used for transfection of HEK-293F cells using 293fectin Transfection Reagent (Thermo Fisher Scientific) according manufactures’ recommendations. Expression of BChE variants was carried out in FreeStyle 293-F Cells media (Thermo Fisher Scientific) in 250 ml culture flasks for 5 days. The cultural media was collected, concentrated using 30 kDa cut-off filter (Millipore) and further purified using TALON Metal Affinity Resin (Clontech) according manufactures’ recommendations.

Zlobin A., Mokrushina Y., Terekhov S., Zalevsky A., Bobik T., Stepanova A., Aliseychik M., Kartseva O., Panteleev S., Golovin A., Belogurov A J.r., Gabibov A, & Smirnov I. (2018). QM/MM Description of Newly Selected Catalytic Bioscavengers Against Organophosphorus Compounds Revealed Reactivation Stimulus Mediated by Histidine Residue in the Acyl-Binding Loop. Frontiers in Pharmacology, 9, 834.

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Luciferase Assay for immRNA Transfection

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HEK-293T MX1P-luc cells were seeded into white 96-well plates at a density of 2.5 × 10⁴ cells per well and incubated overnight. immRNA was diluted to the appropriate concentrations and transfected with 293fectin transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Cells were incubated for 24 h and then lysed and analyzed using the Bright-Glo luciferase assay system (Promega) on a GloMax-Multi microplate reader (Promega) according to the manufacturer’s instructions.

Ho V., Yong H.Y., Chevrier M., Narang V., Lum J., Toh Y.X., Lee B., Chen J., Tan E.Y., Luo D, & Fink K. (2019). RIG-I Activation by a Designer Short RNA Ligand Protects Human Immune Cells against Dengue Virus Infection without Causing Cytotoxicity. Journal of Virology, 93(14), e00102-19.

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SARS-CoV-2 RBD Protein Purification

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A SARS-CoV-2 RBD construct containing a His-tag and Avi-tag was generated, as previously described (51 (link)). Residues 319–541 of the S protein were codon optimized with the N-terminal of signal peptide (MFVFLVLLPLVSSQ) and C-terminal of 6-His tag and Avi-tag (GLNDIFEAQKIEWHE). The DNA encoding sequence was cloned into the mammalian cell expression vector pCAGGS and confirmed by sequencing, before transient transfection in FreeStyle 293-F cells with 293fectin transfection reagent (Thermo Fisher). Culture supernatants were harvested at 5 d after transfection, filtered, and purified by in-house packed affinity purification column with cOmplete His-tag purification resin (Roche). Elutes were buffer exchanged with phosphate-buffered saline (PBS) and concentrated with an Amicon Ultra 10 kDa molecular weight cutoff concentrator (Millipore). Biotinylation was performed with a BirA biotin-protein ligase standard reaction kit (Avidity), according to the manufacturer’s instructions. Excess biotin was removed by five buffer exchanges with an Ultra 10K concentrator (Amicon).

Kardava L., Rachmaninoff N., Lau W.W., Buckner C.M., Trihemasava K., Blazkova J., Lopes de Assis F., Wang W., Zhang X., Wang Y., Chiang C.I., Narpala S., McCormack G.E., Liu C., Seamon C.A., Sneller M.C., O’Connell S., Li Y., McDermott A.B., Chun T.W., Fauci A.S., Tsang J.S, & Moir S. (2022). Early human B cell signatures of the primary antibody response to mRNA vaccination. Proceedings of the National Academy of Sciences of the United States of America, 119(28), e2204607119.

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Influenza HA4900 Protein Expression and Purification

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The recombinant proteins expressed in E. coli BL21 (DE3) RIPL cells were purified according to previously described protocols (10 (link), 12 (link)). Influenza HA4900 was purified from HEK293F suspension cells (Thermo Fisher Scientific, MA) according to previous reports (31 (link)). Briefly, the pAAV-ITRs-HA4900 plasmid was transiently transduced into HEK293F cells maintained in FreeStyle 293 expression medium (Thermo Fisher Scientific, MA) using the 293fectin Transfection Reagent (Thermo Fisher Scientific, MA). The cells were then incubated at 37°C and 8% CO₂ while shaking at 130 rpm overnight. After 12 hours, an equal volume of fresh medium supplemented with sodium butyrate solution (enhancing protein expression; 2 nM final concentration) (Sigma-Aldrich, MO) was added to the cells. On day 7, the culture supernatants were harvested by centrifugation and filtered over 0.22-μm filters (Sartorius Stedim Biotech, Germany). Proteins were purified using HisTrap HP and gel filtration columns (GE Healthcare, IL).

Zhu J., Tao P., Mahalingam M., Sha J., Kilgore P., Chopra A.K, & Rao V. (2019). A prokaryotic-eukaryotic hybrid viral vector for delivery of large cargos of genes and proteins into human cells. Science Advances, 5(8), eaax0064.

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Biotinylation of SARS-CoV-2 RBD Probes

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RBD probes for detection of RBD-specific B cells were obtained by transfection of Expi293 cells with plasmids expressing each RBD variant using the 293fectin transfection reagent (Thermo Fisher Scientific). At day 5 after transfection, the cell supernatants were clarified by centrifugation and filtered. The RBD probes were purified from the supernatant by HisTrap FF column (Cytiva) and HiLoad 16/600 Superdex 200 column (Cytiva) using the AKTA go (Cytiva). The purified RBD probes were biotinylated using a biotin-protein ligase kit (Avidity, Aurora, CO, USA). For flow cytometric analysis, the biotinylated RBD probes were used^{28 (link)}.

Komori M., Nogimori T., Morey A.L., Sekida T., Ishimoto K., Hassett M.R., Masuta Y., Ode H., Tamura T., Suzuki R., Alexander J., Kido Y., Matsuda K., Fukuhara T., Iwatani Y., Yamamoto T., Smith J.F, & Akahata W. (2023). saRNA vaccine expressing membrane-anchored RBD elicits broad and durable immunity against SARS-CoV-2 variants of concern. Nature Communications, 14, 2810.

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