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MembranePro™ Functional Protein Expression Kit - FAQs

View additional product information for MembranePro™ Functional Protein Expression Kit - FAQs (A11667)

9 product FAQs found

I had a problem growing up the 293T cells provided in the MembranePro Functional Protein Expression Kit. Can I use a different cell line?

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.

Your MembranePro Functional Protein Expression Kit/Expi293 MembranePro Expression System does not include a positive control. How do I tell if MembranePro particles were formed?

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.

A GPCR has 2 polypeptides - a ligand-binding polypeptide (receptor chain) and the G-protein (signal transduction chain). Can I use a G-protein coupling receptor (RG-form) specific ligand as a tracer?

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.

What is the size range of proteins produced with the MembranePro Functional Protein Expression Kit and the Expi293 MembranePro Expression System?

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.

Does the expression vector in the MembranePro Functional Protein Expression Kit offer a His tag?

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.

Can I use an existing stable cell line expressing a GPCR of interest with the MembranePro Functional Protein Support Kit?

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.

Can I use a different expression vector with the MembranePro Functional Protein Expression Kit or the Expi293 MembranePro Expression System?

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.

Is there a positive control included with the MembranePro Functional Protein Expression Kit or the Expi293 MembranePro Expression System?

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.

I am interested in expressing some functional membrane proteins in mammalian cells. Do you offer a system that will allow me to do this?

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.