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查看更多产品信息 Expi293™ MembranePro™ Expression System - FAQs (A25869, A25870)
36 个常见问题解答
尽管您可测试并使用其它细胞系，不过我们还是推荐您使用Expi293F细胞，因为该试剂盒的性能（即,使用ExpiFectamine转染试剂所获得的转染效率）曾针对这一细胞系进行过专门的优化。
尽管您可测试其他的表达载体和启动子，不过我们还是建议使用pEF6 V5-His TOPO TA表达载体来优化蛋白得率，因为这些试剂盒的性能曾针对包含EF-1α启动子的表达载体进行过专门优化。
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。
尽管您可测试其他的表达载体和启动子，不过我们还是建议使用pEF6 V5-His TOPO TA表达载体以获得最佳的产量，因为该试剂盒的性能针对这一包含EF-1α启动子的表达载体进行过专门优化。
尽管您可测试其他细胞系，不过我们还是推荐您使用Expi293F细胞，因为该试剂盒的性能（即使用ExpiFectamine转染试剂所获得的转染效率）针对这一细胞系进行了专门的优化。
您可于离心后通过肉眼观察管底沉积来判断MembranePro颗粒是否形成。您也可通过受体-配体结合实验来测试MembranePro颗粒的功能。
除非您能够解离MembranePro颗粒，否则gag蛋白无法被除去。我们建议您使用膜蛋白分离的方法，减少gag。本系统得到的定量分析的蛋白中，每次反应得到50-500 μg总蛋白，您的GPCR只占较小部分。
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。
我们表达和测试过50kDa左右的GPCR蛋白。在理论上，对于蛋白的胞外域或跨膜域没有大小限制。不过，如果C-端结构域过大，则可能会存在包装方面的问题。
尽管您可测试其他细胞系，不过我们还是推荐您使用Expi293F细胞，因为该试剂盒的性能（即使用ExpiFectamine转染试剂所获得的转染效率）针对这一细胞系进行过专门的优化。
尽管您可测试其他的表达载体和启动子，不过我们还是建议使用pEF6 V5-His TOPO TA表达载体，因为该试剂盒的性能针对这一包含EF-1α启动子的表达载体进行过专门优化。
不包含。不过，您可于离心后通过肉眼观察管底沉积来判断MembranePro颗粒是否形成。您也可通过受体-配体结合实验来测试MembranePro颗粒的功能。
如需在Exp293F细胞中高产量表达功能性的膜蛋白，我们还准备了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量大大提升，超出标准贴壁培养MembranePro功能性蛋白表达系统—产品的表达量20倍以上。
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。
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。
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。
While other cell lines can be tested, we recommend using the Expi293F cells, as the performance of the kit has been optimized for use with this cell line (i.e., transfection efficiency using ExpiFectamine Transfection Reagent).
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
While other expression vectors and promoters can be tested, we recommend using the pEF6 V5-His TOPO TA expression vector for optimal yields, as the performance of these kits has been optimized for use with this expression vector containing the EF-1alpha promoter.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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.
While other expression vectors and promoters can be tested, we recommend using the pEF6 V5-His TOPO TA expression vector for optimal yields, as the performance of the kit has been optimized for use with this expression vector containing the EF-1alpha promoter.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
While other cell lines can be tested, we recommend using the Expi293F cells, as the performance of the kit has been optimized for use with this cell line (i.e., transfection efficiency using ExpiFectamine Transfection Reagent).
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
You can visually tell if MembranePro particles formed via a pellet at the bottom of the tube following precipitation. You can also test function of your MembranePro particles via receptor-ligand binding studies.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The gag protein can't be removed unless you dissociate the MembranePro particles. Our recommendation is that methods used to isolate membrane proteins should eliminate gag. This system makes analytical quantities of protein, 50-500 µg total protein per reaction, of which a small fraction is your GPCR.
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.
The GPCRs we produced and tested are approximately 50 kDa. In theory, there is no limit on the size of the extracellular or transmembrane domains. However, there may be packaging issues if the C-terminal domain is too large.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
While other cell lines can be tested, we recommend using the Expi293F cells, as the performance of the kit has been optimized for use with this cell line (i.e., transfection efficiency using ExpiFectamine Transfection Reagent).
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
While other expression vectors and promoters can be tested, we recommend using the pEF6 V5-His TOPO TA expression vector, as the performance of the kit has been optimized for use with this expression vector containing the EF-1alpha promoter.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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.
For high-yield expression of functional membrane proteins in Expi293F 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.
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.
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.
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 do not need a license to use the Expi293 MembranePro Expression System. This product is "For Research Use Only. Not for use in diagnostic procedures."
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.