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查看更多产品信息 MembranePro™ Functional Protein Expression Kit - FAQs (A11667)
18 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试剂盒自带的293FT细胞，因为该试剂盒的性能（即,转染效率）曾针对这一细胞系进行过专门的优化。
您可于离心后通过肉眼观察管底沉淀的MembranePro颗粒。您也可通过受体-配体结合实验来测试MembranePro颗粒的功能。
如果配体结合链是被单独转染的，则可能无法与G蛋白相偶联。我们证实了激动剂对MembranePro颗粒所捕获5HT1a和毒蕈碱M1受体的配体结合效果。基于亲和力的差异，不同的激动剂竞争性地占据了MembranePro颗粒上的全部5HT1a和毒蕈碱M1受体，这提示MembranePro颗粒所捕获受体仍能胜任生理性的相互作用。在直接结合研究中，激动剂以低亲和力形式（更高的Kd值）进行结合，提示G蛋白极少或未能与受体相偶联。不过，这一结合仍是特异性和竞争性的。您可通过共转同源的G蛋白来增加结合效力。
我们表达和测试的GPCR在50 kDa左右。理论上，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，则可能会遇到包装方面的问题。
pEF6 TOPO TA载体的C端附加了His标签，这一位点不具有功能性作用。只有当您构建的序列中不含终止密码子时，His标签才会表达并用于纯化操作。本载体的N端不含His标签。
理论上这是可行的，但我们未就这一方法进行过验证。假设细胞系已经表达了足够的GPCR蛋白，且通过了传统的膜蛋白制备成功进行了功能验证（即，受体-配体结合实验），您可使用MembranePro功能性蛋白支持试剂盒（货号A11668，A11669，A11670）来生成MembranePro病毒颗粒。GPCR的蛋白得率主要基于稳转细胞系的表达水平。
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。
While other cell lines can be tested, we recommend using the supplied 293FT cells, as the performance of the kit has been optimized for use with this cell line (i.e., transfection efficiency).
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 the function of your MembranePro particles via receptor-ligand binding studies.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
If a ligand-binding chain is transfected alone, there may not be coupling of G-protein. We have demonstrated agonist ligand binding to 5HT1a and muscarinic M1 receptors captured on MembranePro particles. Different agonists compete ligand off both muscarinic and 5HT1a receptors in MembranePro particles, and they do so in order of affinity, indicating physiologically relevant interaction with the receptors on MembranePro particles. In direct binding studies, agonist binds in the low-affinity form (higher Kd), indicating little or no G-protein is coupled to the receptor. However, the binding is still specific and competitive. You may see increased binding by coexpressing the cognate G-protein.
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.
The MembranePro Functional Protein Expression System is optimized for use with the pEF6 vector. However, cloning your gene into the pEF6/V5-His-TOPO vector without a stop codon and in frame with the polylinker will result in a fusion protein with V5 and polyhistidine (6×His) tags on the C-terminus of your protein. As the C-terminus of your transmembrane protein will likely be inside the VLP, these tags will be inaccessible to purification resins and antibodies. In theory, these tags could be used to identify and isolate a fusion membrane protein after denaturing the VLP; however, the MembranePro Functional Protein Expression System does not support using the tags for extraction and purification.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Theoretically this is possible, but we have not validated this approach. You should be able to generate MembranePro particles using the MembranePro Functional Protein Support Kits (Cat. Nos. A11668, A11669, A11670) assuming the cell line has adequate GPCR expression, and functional testing (i.e., receptor-ligand binding studies) with conventional membrane preps from these cell lines was successful. The protein yield for the GPCR will depend on the expression levels from their stable cell line.
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.