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View additional product information for Expi293F™ Cells - FAQs (A14528, A14527)
18 product FAQs found
未含。不过，您可于离心后通过肉眼观察管底沉积的MembranePro病毒颗粒。您也可通过受体-配体结合研究来测试MembranePro病毒颗粒的功能。
我们提供了MembranePro功能性蛋白表达系统，来用于哺乳动物细胞膜表面蛋白——包括G蛋白偶联受体（GPCR）——以水溶性形式的表达和展示。这一系统使用病毒样颗粒来捕捉细胞膜表面的脂筏区域，因为VLP会从细胞的这一区域分泌出来。通过使用这一系统，用户可将内源性或过表达的GPCR及其它细胞膜表面蛋白在其天然环境下捕获和展示，再将其用于下游分析。由于VLP是由细胞完成包装并分泌至培养基中的，因此用户可通过简单地倒出和澄清培养基，再沉淀分离VLP，来实现功能性膜蛋白的分离。相比制备细胞膜组份的操作而言，这一实验能大大节省时间，精力，并降低设备需求。由于VLP能够捕获细胞膜表面的受体富集区，您的GPCR相比细胞膜的粗提操作得到了大幅的富集。
MembranePro功能性蛋白表达系统有两种标准的产品配置：MembranePro功能性蛋白表达试剂盒（Functional Protein Expression Kit，货号A11667），以便利的完整试剂盒形式提供，包含了10次反应的所需的全部试剂；MembranePro功能性蛋白支持试剂盒（Functional Protein Support Kit），包含膜蛋白功能性表达所需的相关试剂，但未含293FT宿主细胞或表达载体克隆模块。MembranePro功能性蛋白支持试剂盒有三种规格：10次，60次或600次反应（货号分别为A11668、A11669、A11670）。
如需在Exp293F细胞中高表达功能性膜蛋白，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效率大大提升，进而超出常规贴壁培养的MembranePro功能性蛋白表达系统的表达效率20倍以上。请参见应用说明了解更多详情。
注意：Expi293F细胞（货号A14527）与pEF6 V5 His TOPO表达载体试剂盒（货号K961020）未包含于Expi293 MembranePro表达系统中，因此需单独购买。
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。
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。
No. However, you can visually tell if MembranePro particles formed via a pellet at the bottom of the tube following precipitation. You can also test the function of your MembranePro particles via receptor-ligand binding studies.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We offer the MembranePro Functional Protein Expression System for the expression and display of mammalian cell surface membrane proteins, including G-protein coupled receptors (GPCRs), in an aqueous-soluble format. The system uses virus-like particles (VLPs) to capture lipid raft regions of the cell’s plasma membrane as the VLPs are secreted from the cell. Using this system, it is possible to capture and display endogenous or overexpressed GPCRs and other cell surface membrane proteins in their native context for downstream assays. Since the VLPs are packaged by the cell and secreted into the culture medium, VLPs allow for the isolation of functional membrane proteins by simply decanting and clarifying the culture medium, and isolating the VLPs by precipitation. This represents a substantial savings in time, effort, and required machinery over preparing cell membrane fractions. Because VLPs capture receptor-rich regions of the plasma membrane, your GPCR may also be substantially enriched over crude membrane preparations.
The standard MembranePro Functional Protein Expression System is offered in two configurations: The MembranePro Functional Protein Expression Kit (Cat. No. A11667), which provides the convenience of a complete kit with all the reagents supplied for 10 reactions, and the MembranePro Functional Protein Support Kit, which includes the reagents for functional expression of membrane proteins but does not contain the expression vector cloning module or the 293FT host cells. The MembranePro Functional Protein Support Kit is offered in three sizes, allowing 10, 60, or 600 reactions (Cat. Nos. A11668, A11669, A11670, respectively).
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 for more details.
Note: Expi293F cells (Cat. No. A14527) and pEF6 V5 His TOPO Expression Vector Kit (Cat. No. K961020) are not supplied with the Expi293 MembranePro Expression System and have to be purchased separately as required.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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.
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.
Expi293F Cells is classified biosafety level 2.
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No, they do not need to be spun down to remove the DMSO. As the cells (1 mL) will be transferred to a shaker flask containing 30 mL of pre-warmed expression medium, the DMSO will be diluted so that it won't affect the cells.
Please see the following application note "Creation and Scale up of a Stable Cell Line using ExpiCHO Products" (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017764_CreatScaleup_StableCellExpiCHO_UB.pdf), that presents our suggested protocol for generation of a stable cell line from ExpiCHO-S cells, which like Expi293F, are grown in suspension.
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.
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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.
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.