His-mATGL and its point mutants were recombinantly expressed in Expi293F™ cells (Thermo Fisher Scientific, Waltham, USA). The cells were cultivated in Expi Expression Medium (Thermo Fisher Scientific, Waltham, USA) at standard conditions (37°C, 95% humidified atmosphere, 7% CO2). Cell density was determined in a CASY Cell Counter and Analyzer System (OMNI Life Science, Bremen, Germany). Cells in a total culture volume of 10 ml were transfected with 10 μg of plasmid DNA using the ExpiFectamineTM 293 Transfection Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Two days after transfection, cells were harvested by centrifugation at 500 g and 4°C for 5 min. The cell pellet was washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4). Cells were disrupted immediately after harvesting by sonication (Sonopuls GM3100 equipped with a MS-72 tip; Bandelin, Berlin, Germany) in sucrose solution (250 mM sucrose, 1 mM EDTA, 1 mM DTT, 20 μg/ml leupeptin, 2 μg/ml antipain, and 1 μg/ml pepstatin) on ice. The homogenate was centrifuged at 1,000 g and 4°C for 20 min. The postnuclear fraction was collected and protein concentration was determined by the Bradford protein assay (Bio-Rad Laboratories, Hercules, USA) using (BSA) as standard.
Expifectaminetm 293 transfection kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ExpiFectamine™ 293 Transfection Kit is a reagent-based system designed for the transient transfection of mammalian cells. It facilitates the efficient delivery of DNA, RNA, or other macromolecules into a variety of cell types, enabling the expression of the desired genetic material.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Expi293f cells, by Thermo Fisher Scientific (3 mentions)
Expi293ftm cells, by Thermo Fisher Scientific (2 mentions)
Kta go purification system, by GE Healthcare (2 mentions)
Hitrap protein g hp column, by GE Healthcare (2 mentions)
Expi expression medium, by Thermo Fisher Scientific (1 mentions)
Sonopuls gm 3100, by Bandelin (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Hitraptm desalting column, by GE Healthcare (1 mentions)
Strep tactin xt, by GE Healthcare (1 mentions)
Histrap hp, by GE Healthcare (1 mentions)
Zymopure 2 plasmid maxiprep kit, by Zymo Research (1 mentions)
Slide a lyzer dialysis cassette, by Thermo Fisher Scientific (1 mentions)
Expi293, by Thermo Fisher Scientific (1 mentions)
Na934, by Cytiva (1 mentions)
Plasmid purification kit, by Qiagen (1 mentions)
Ez fusion ht cloning kit, by Enzynomics (1 mentions)
10 protocols using expifectaminetm 293 transfection kit
1
Recombinant Expression of His-mATGL Mutants
2
Reformatting and Purification of Chimeric Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The reformatting, expression, and purification of full-length antibodies was performed as described previously [31 (link),35 (link)]. Isolated yeast vectors were sequenced, and the VL fragment was reformatted into pTT5-derived vectors by golden gate assembly. For the soluble expression of full-length chimeric antibodies, Expi293FTM cells (Thermo Fisher Scientific) were transiently transfected using the ExpiFectamineTM 293 Transfection Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. For purification, cell culture supernatants were collected five days post transfection, sterile-filtered, and applied to a MabSelectTM PrismA HP column (GE Healthcare, Piscataway, NJ, USA) using an ÄKTA pureTM chromatography system (GE Healthcare). One-armed molecules were captured by IMAC (HisTrap HP, GE Healthcare), followed by Strep-Tactin XT affinity chromatography according to the manufacturer’s protocol. Buffer exchange against PBS was performed using a HiTrapTM Desalting column (GE Healthcare).
3
SARS-CoV-2 Spike Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
pAd/SARS-CoV-2-S1WU and pAd/SARS-CoV-2-SAS1SA were amplified and purified using ZymoPURE II plasmid maxiprep kit (Zymo Research). For Expi293 cell transfection, we used ExpiFectamineTM 293 Transfection Kit (ThermoFisher) and followed the manufacturer’s instructions. Cells were seeded 3.0 × 106 cells/ml 1 day before transfection and grown to 4.5–5.5 × 106 cells/ml. One microgram of DNA and ExpiFectamine mixtures per 1 ml culture were combined and incubated for 15 minutes before adding into 3.0 × 106 cells/ml culture. At 20 hours post-transfection, enhancer mixture was added, and culture was shifted to 32°C. The supernatants were harvested 5 days post transfection and clarified by centrifugation to remove cells, filtered through 0.8 μm, 0.45 μm, and 0.22 μm filters, and either subjected to further purification or stored at 4°C before purification.
4
Transient BiTE Expression in Expi293F Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sorted BiTE candidates assembled into expression plasmids were then transfected into 1 mL Expi293FTM cells (#A14527, ThermoFisher Scientific) using ExpifectamineTM 293 Transfection kit (#A14524, ThermoFisher Scientific) as per manufacturers protocol in 96-well 2-mL culture plates. Seventy-two hours after transfection, BiTE supernatant was collected and used for bulk reporter activation assay.
5
Purification of Monoclonal Antibodies from Expi293F Cells
6
Recombinant Antibody Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibody heavy and light chain sequences were appended with restriction sites for insertion into human IgG1 antibody expression vectors (42 (link)). For each antibody, Expi293F cells were co-transfected with heavy and light chain plasmids using the ExpiFectamineTM 293 Transfection Kit (Thermo Fisher Scientific, Massachusetts, USA). Secreted antibody in supernatant from transient transfection were purified with Protein G or A resin (GenScript, New Jersey, USA) and concentrated using an Amicon Ultra-4 Centrifugal 30K Filter Unit (MilliporeSigma, Maryland, USA), then stored at 4°C.
7
Purification of Recombinant Monoclonal Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expi293F cells (Thermo Fisher Scientific) were transfected with plasmids carrying J08 and 02M04Ab heavy and the light chains with a 1:2 ratio using ExpiFectamineTM293 Transfection Kit (Thermo Fisher Scientific) as recommended by the manufacturer. 3 and 6 d after transfection, cell cultures were harvested and clarified by centrifugation (1,100 ×g for 10 min at room temperature). Supernatants were recovered and filtered with a 0.45-μm filter to remove particulate material. The purification process was conducted at room temperature using the ÄKTA Go purification system (GE Healthcare Life Sciences) through a 1 ml HiTrap Protein G HP column (GE Healthcare Life Sciences), previously equilibrated with a loading buffer (0.02 M NaH2PO4 pH 7). Each mAb was eluted from the column in 1-ml fractions of elution buffer (0.1 M glycine-HCl, pH 2.7) and collected in vials predispensed with 100 µl of neutralization buffer (Tris-HCl pH 9.0). Protein-containing fractions were pooled and dialyzed in PBS 1× Buffer pH 7.4 with a 1:200 ratio using Slide-A-Lyzer Dialysis Cassettes, 10 K MWCO, 3 ml (Thermo Fisher Scientific) at 4°C overnight. The final Ab concentration was determined by measuring the absorbance at 562 nm using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Purified Abs were stored at −80°C prior to use.
8
Transient Expression of Human ACE2 Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human ACE2 IgG and IgM fusion proteins were obtained from transient transfection using the Expi293TM system (Thermo Fisher Scientific). All recombinant pD2610-v10 plasmids used in this study were generated by ATUM and transient transfection was carried out following the manufacturer’s protocol for the ExpiFectamineTM 293 transfection kit (Thermo Fisher Scientific). Four days after transfection, the supernatant was collected after centrifugation of the culture and filtered through a 0.2 µm filter to remove cells and debris. All purifications were performed using mixed-mode and anion-exchange chromatography as described previously [29 (link)].
9
ATGL Variants Expression and Triglyceride Hydrolase Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ATGL variants were expressed in Expi293 (Thermo Fisher Scientific, A14635) cells. 2.5 × 106 cells were transfected with 10.8 µg DNA using the ExpiFectamineTM 293 Transfection Kit (Thermo Fisher Scientific, A14525) according to manufacturer’s instructions. Cells were harvested after 48 h, washed with PBS, resuspended in 4 ml HSL buffer (0.25 M sucrose, 1 mM EDTA, 1 mM DTT, 1 µg/ml pepstatin, 2 µg/ml antipain and 20 µg/ml leupeptin) and lysed by ultra-sonification (20% amplitude, 30 s). After centrifugation (20,000 × g, 4 °C for 10 min), the soluble extract was adjusted to a total protein level of 3.5 µg/µl. Protein expression (5 µg per lane) was validated by western blotting on PVDF using Abs against Protein A (1:3000, anti-rabbit-IgG-HRP-linked, NA934, GE Healthcare/Amersham) or GAPDH (1:10000, #2118 S, Cell signaling technology) and HRP-linked secondary antibody: anti-rabbit (1:10000, #7074, CST). The in vitro radiolabeled triglyceride hydrolase activity assay was performed according to Schweiger et al.52 (link).
10
Expression and Purification of Atezolizumab-VEGF-Grab Fusion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genes encoding the scFv of atezolizumab were cloned into the N-terminal of VEGF-Grab in the pcDNA3.1 vector using the EZ-Fusion™ HT Cloning Kit (Enzynomics, EZ015TS). Each cloned pcDNA 3.1 vector was amplified using DH5-alpha competent cells (Enzynomics, CP010) and purified using a plasmid purification kit (Qiagen, 12145). Purified vectors were transfected into Expi293FTM cells using the ExpiFectamineTM 293 transfection kit (Thermo, A14525). Transfected cells were cultured for 4 days in the presence of enhancers 1 and 2. VEGF-Grab or Ate-Grab in the supernatant were bound to Protein A resin and washed with PBS at 10 times the resin volume. Subsequently, they were neutralized with 1 M Tris-HCL (pH 8.0) and 250 mM NaCl, eluted with 100 mM glycine and 250 mM NaCl, pH 2.7, and purified via size exclusion chromatography.
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!