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View additional product information for Expi293™ Expression System Kit - FAQs (A14635)
43 product FAQs found
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。
请参见应用说明(https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/CellCultureandTransfection/pdfs/Expi293Adaption-MembraneProTech_AppNoteGlobal_FHR.pdf)关于使用MembranePro试剂盒适应Expi293系统的具体实验方案。
请参见应用说明(https://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.0358.file.dat/PG1391-PJ6257-CO28716-Expi293-10L-Wave-Bag-App-Note-Global_FHR.pdf)中将Expi293表达系统适应于WAVE Bioreactor系统的操作方法。
可以，请参见Expi293表达系统96孔微量滴定板的应用说明(https://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.28891.file.dat/expi293-microtiter-plates-co25793.pdf)。简而言之，您可在2 毫升反应孔模块中使用700 微升的培养体系，并在3 毫米throw的涡旋振荡器（如Eppendorf MixMate混合器）上以1250-1500 rpm的转速进行振荡培养。
请参考应用说明(https://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.61153.file.dat/expi293-protein-yield-co25763.pdf)获取在Expi293表达系统中优化蛋白产量的相关提示。
我们推荐用户单独将抗体的重链和轻链亚基克隆至pcDNA3.4 TOPO载体中，之后再优化两种质粒的转染比例。每一种抗体均需要调整重链:轻链的使用比例，以获得最佳的抗体表达效果。将两条链克隆在各自的载体上使这一调整变得更容易。使用相同的载体可确保一开始每一亚基的表达处于同一水平。
对于1L的培养体积，我们内部的科学家使用3L带挡板的培养瓶在转速80-85 rpm进行过培养；其使用的摇床为Innova 0.75英寸轨道摇床。
在使用手册中，我们为不同型号的培养瓶提供了最佳的振荡速度。对绝大多数培养瓶而言，使用过快或过慢的振荡速度会引起细胞剪切力过大或者通气不足，从而导致表达水平的显著下降。这一负面效应在培养板中表现得更突出，因为在孔板中静态与动态液流之间的转变更剧烈。
这要视所使用的培养瓶类型而定，不过常规经验是使用培养瓶的1/4体积。
不需要。Expi293表达系统经设计省略了培养基的换液操作。因此无需在转染操作后去除转染复合物或更换培养基。
新制备的转染复合物所获得的转染效率最佳。不过，这些复合物可在至少一小时之内保持稳定。
Expi293F细胞的生长和表达属性决定了在转染7天之后，培养基将临近耗竭，并应该已经达到最高蛋白表达量。继续孵育将导致培养细胞活力的显著下降。
最佳的表达时间随不同的目的蛋白而发生变化，但大多数测试蛋白的最佳表达时间窗为3-7天。由于本系统具有良好的扩展性，我们推荐您可在放大扩种之前进行一个小规模测试实验，来决定何时收获目的蛋白。
尽管Expi293培养基能够支持更高的细胞密度，但我们并不推荐以超出5–6 x 10e6个细胞/毫升的密度来培养Expi293细胞，因为这会降低后续的转染和蛋白表达效率。在更高的细胞密度时，遇到培养活力降低临界点的可能性也会增加。如果您培养的细胞超出5–6 x 10e6个细胞/毫升，请按照手册中的描述传代一至两次，监控它们的活力和生长速率。使用蛋白表达对照IgG或得率已知的表达载体来测试转染效果，从而确定细胞表达性能是否受到影响。
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。
Most 293 cell lines can be adapted directly from conventional serum-containing or other serum-free media into Expi293 Expression Medium by either using the Direct Adaptation method or Sequential Adaptation method, described in the manual.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Please refer to the Application Note (http://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/CellCultureandTransfection/pdfs/Expi293Adaption-MembraneProTech_AppNoteGlobal_FHR.pdf) for a protocol for adapting the MembranePro kit for use with the Expi293 System.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Please see the Application Note (http://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.0358.file.dat/PG1391-PJ6257-CO28716-Expi293-10L-Wave-Bag-App-Note-Global_FHR.pdf) for a method for the adaptation of the Expi293 Expression System for use in the WAVE Bioreactor System.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Yes, see the Application Note (http://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.28891.file.dat/expi293-microtiter-plates-co25793.pdf) on using 96-well microtiter plates with the Expi293 Expression System.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Please refer to the Application Note for tips on optimizing protein yield using the Expi293 Expression System (http://www.thermofisher.com/content/dam/LifeTech/migration/files/proteins-expression-isolation-analysis/pdfs.par.61153.file.dat/expi293-protein-yield-co25763.pdf).
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We recommend cloning the heavy and light chain subunits separately into the pcDNA3.4 TOPO vector and then optimizing the ratios of the 2 plasmids. Each antibody needs to have the ratio of heavy to light chain adjusted for optimal antibody expression. Having the chains on separate plasmids makes this easier. Using the same plasmid will also ensure equal levels of expression of each subunit to start with.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Our in-house scientists have cultured a 1 L volume in a 3 L baffled shake flask at 80-85 rpm. This was done using an Innova shaker with a 0.75-inch orbital throw.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
In the manual, we have provided optimal shake speeds for various formats. For most flasks, there would be a drop-off in expression when you go too fast or too slow because of cell shear stress or insufficient aeration. The effect is sharper in plates, where there is a sharper transition between static and moving fluid in wells.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
This depends on the flask, but a general rule of thumb is to use one quarter the volume of the flask.
Find additional tips, troubleshooting help, and resources within our Protein Expression 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.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
The best transfection efficiencies are obtained when transfection complexes are used fresh. However, they should be stable for at least an hour.
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The growth and expression characteristics of Expi293F cells are such that, by 7 days post-transfection, the culture medium should be close to being spent and maximal protein expression should have already been achieved. Continued incubation will result in a large decrease in cell culture viability.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The optimal expression time will be different for each protein, but most tested proteins fall within a 3-7 day window. As the system scales well, it is recommended 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 Protein Expression Support Center.
While the Expi293 Expression Medium can support much higher cell densities, we do not recommend growing your Expi293F 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 them 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 an expression construct of known yield to determine if cell expression performance has been impacted.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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.
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.
The formation of intact IgG molecules may be quantified using a sandwich ELISA designed to capture and detect rabbit IgG. Besides the rabbit IgG positive control vector, 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 (H+L) Cross-Adsorbed Secondary Antibody, HRP (Cat. No. A10547), Protein A Coated Plates, Clear, 96-Well (Cat. No. 15130), TMB Substrate Kit (Cat. No. 34021), SuperBlock (TBS) Blocking Buffer (Cat. No. 37535), and PBS or TBS buffer for washes. There is an example procedure in our Protein A Coated Plates manual (https://tools.thermofisher.com/content/sfs/manuals/MAN0011310_Thermo Scientific_ProteinA_G_AG_Coat_96Well_UG.pdf). Please note that 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.
We offer pRABBIT IgG IRES-EmGFP Positive Control Vector, Cat. No. A39243, which you can use to monitor your transfection and expression.
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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.
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Expi293F cells must be recovered from freezing and passaged at least 3 times using the procedure outlined in the manual to ensure optimal performance. Cells should maintain performance for at least 30 passages if maintained in accordance with the protocol in the manual.
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.