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View additional product information for Expi293™ Expression Medium - FAQs (A1435102, A1435101, A1435104, A1435103)
20 product FAQs found
Expi293表达培养基并不直接兼容ProBond或Ni-NTA纯化系统。我们推荐在用His标签的纯化操作之前进行一次缓冲体系交换或样本透析。
大多数293细胞系均能适应从传统含血清或其它无血清培养基向Expi293表达培养基中的切换按照手册中所描述的直接切换法或序贯切换法来操作，大多数293细胞系均能适应从传统含血清或其它无血清培养基向Expi293表达培养基中的切换。 注意：切换后的细胞可能无法达到与Expi293F细胞同等的高水平表达。
我们推荐使用pcDNA 3.4-TOPO TA载体（货号A14697）。这一载体含有天然的全长CMV启动子和克隆位点下游的WPRE（旱獭转录后调控元件），这两个元件均有利于基因的高水平表达（表达比pcDNA3.3 TOPO载体高大约2-3倍，而后者又比标准的pcDNA载体高2-5倍）。当然，实际表达水平还要视具体蛋白而定。
除了Opti-MEM I低血清培养基为无血清但不是非动物源性的外，本系统中的其余成份均为非动物源性的，请参考应用说明(https://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.5943.file.dat/expi-293-animal-origin-free-co25751.pdf): 在非动物源性的条件下使用Expi293表达系统。
我们为蛋白的稳定高产量生产提供Freedom CHO-S与Freedom DG44试剂盒。我们为瞬时高产量生产提供ExpiCHO表达系统，Expi293表达系统，FreeStyle 293 表达系统，FreeStyle Max 293表达系统和FreeStyle Max CHO表达系统。如需在Exp293F细胞中高效表达功能性膜蛋白，我们还提供了Expi293 MembranePro 表达系统（货号A25869，A25870），这款产品将Expi293的扩展性/易用性与MembranePro技术整合起来，能够将膜蛋白的表达产量大大提升，超出标准贴壁培养的MembranePro功能性蛋白表达系统的表达产量20倍以上。请参见 应用说明(https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/ProteinExpressionAnalysis/pdfs/PG1391-PJ6257-CO28704-Expi293Adaption-MembraneProTech_AppNoteGlobal-FHR.pdf) 以了解更多详情。
Expi293 Expression Medium is not directly compatible with ProBond or Ni-NTA purification systems. We recommend performing a buffer exchange or dialyzing the samples before His-tag purification.
Other 293 cell lines may be used with the Expi293 Expression System. However, before these cell lines may be used for transfection studies, they must be adapted to serum-free, suspension culture in Expi293 Expression Medium and evaluated for transfection and expression.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We recommend using the pcDNA 3.4-TOPO TA vector (Cat. No. A14697). This vector contains the native, full-length CMV promoter and a WPRE (Woodchuck Posttranscriptional Regulatory Element) downstream of the cloning site, both of which contribute to high level gene expression (about 2-3 fold higher expression than with pcDNA3.3 TOPO vector, which in turn provides 2-5 fold higher expression than with a standard pcDNA vector). Of course, the expression level is also protein-dependent.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
All the components of the system are animal-origin free except for the Opti-MEM I Reduced Serum Medium that is serum-free but not animal-origin free. Please see the Application Note for using the Expi293 Expression System under animal origin-free conditions:
http://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.5943.file.dat/expi-293-animal-origin-free-co25751.pdf
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
For stable high-yield expression, we offer the Freedom CHO-S Kit (Cat. No. A1369601) and Freedom DG44 Kit (Cat. No. A1373701). For transient high-yield expression, we offer the ExpiCHO Expression System (Cat. No. A29133), Expi293 Expression System (Cat. No. A14635), FreeStyle 293 Expression System (Cat. No. K900001), FreeStyle Max 293 Expression System (Cat. No. K900010), and FreeStyle Max CHO Expression System (Cat. No. K900020). For high-yield expression of functional membrane proteins in Exp293F cells, we offer the Expi293 MembranePro Expression System (Cat. Nos. A25869, A25870) that combines the scalability and ease of use of Expi293 and the technology of MembranePro to allow an increase of more than 20-fold in membrane protein yield compared to the standard, adherent culture MembranePro Functional Protein Expression System. Please see the Application Note (http://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/ProteinExpressionAnalysis/pdfs/PG1391-PJ6257-CO28704-Expi293Adaption-MembraneProTech_AppNoteGlobal-FHR.pdf) for more details.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
For most flasks, there is a dropoff in expression when you go too fast or too slow because of cell shear stress or insufficient aeration. The effect is greater in plates where there is a sharper transition between static and moving fluid in the wells.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
No. The Expi293 Expression System is designed to run without media exchanges. There is no need to remove transfection complexes or to change growth medium following transfection.
The optimal expression time will be different for each protein, but most tested proteins fall within a 3-7 day window. Since the system scales well, it is recommended that you run a small-scale pilot experiment to determine when to harvest your protein of interest prior to scaling up.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
While Expi293 medium can support much higher cell densities, we do not recommend growing your Expi293 cultures beyond 5-6 x 10e6 cells/mL as subsequent transfection and protein expression efficiencies may be reduced. At higher densities, there is also the increased possibility of reaching the point of reduced culture viability. If your seed culture does exceed 5-6 x 10e6 cells/mL, passage the cells once or twice as detailed in the manual, monitoring them for viability and growth rate. Perform a test transfection using the Protein Expression control IgG or your own expression construct of known yield to determine if cell expression performance has been impacted.
This product should be stored at 2-8 degrees C and should not be frozen. The biggest issue with accidentally freezing this product is its solubility. If this product was accidentally frozen, we recommend placing it in a 2-8 degree environment and allowing it to slowly thaw overnight. If the product is fully thawed and shows no signs of precipitation, then it should still be usable, but we cannot guarantee effectiveness.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Expi293 Expression Medium does not contain antibiotics. We do not recommend the use of antibiotics, however, 5 mL/L of Antibiotic-Antimycotic (Cat. No. 15240) containing penicillin, streptomycin, and amphotericin B may be used when required, as stated on page 1 in the manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/expi293_exp_medium_man.pdf).
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
You can have Anti-Clumping Agent and Pluronic F-68 in the medium while growing Expi293 cells. You need to remove Anti-Clumping Agent at least 2 passages prior to transfection of Expi293 cells because it interferes with transfection. You can add it back to the medium after transfection. Pluoronic F-68 can be present during transfection as it does not interfere with transfection.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
No. The Expi293 Expression System is designed to run without media changes. There is no need to remove transfection complexes or to change growth medium following transfection.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The Enhancers are designed to work together for maximal expression. Addition of just one Enhancer will result in reduced expression and may be anywhere from one third to two thirds the level of expression obtained if both Enhancers had been added on time.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The formation of intact IgG molecules may be quantitated using a sandwich ELISA designed to capture and detect rabbit IgG. Besides the rabbit IgG positive control, reagents, and consumables that are included in the kit, you will also need purified rabbit IgG to be used as a standard, F(ab')2 goat anti-rabbit IgG HRP conjugate (Cat. No. A10547), Protein A-coated plates (Cat. No. 15130 for clear plates used in colorimetric detection), TMB colorimetric substrate (Cat. No. 34021), SuperBlock (TBS) Blocking Buffer (Cat. No. 37581), and PBS or TBS buffer for washes. There is an example procedure in our Protein A-coated plates manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011310_Pierce_ProteinA_G_AG_Coat_96Well_UG.pdf). Please note, our R&D scientists determine titer values from crude cell culture supernatants using a Pall Life Sciences FortéBio Octet instrument equipped with a protein A biosensor.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.