The Expi293F cells supplied with the Expi293 MembranePro Expression System had poor viability upon thawing. Can I use a different cell line?
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).
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.
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.
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.
I have misplaced the pEF6 V5-His TOPO TA expression vector from the Expi293 MembranePro Expression System. Can I use a different expression vector?
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.
I do not have Expi293F cells. Can I use a different cell line with the Expi293 MembranePro Expression System?
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).
I am using your Expi293 MembranePro Expression System for expression of my membrane protein. Since you do not provide a positive control with the kit, I am concerned that I might not be able to 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 function of your MembranePro particles via receptor-ligand binding studies.
Can the gag protein be removed for downstream structure analysis?
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.
Can I use the Expi293 Expression System for scalable production of membrane proteins?
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.
What is the size range of proteins produced with 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.
Can I use a different cell line with the Expi293 MembranePro Expression System?
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).
Can I use a different expression vector with 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, as the performance of the kit has been optimized for use with this expression vector containing the EF-1alpha promoter.
Is there a positive control included with 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.
I am interested in getting high yield of a functional membrane protein in mammalian cells. Do you offer a system that will allow me to do this?
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.
Can I use a different 293 cell line with the Expi293 Expression System?
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.
Is the Expi293 Expression System completely animal-origin free (AOF)?
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
I am interested in a mammalian expression system that will allow me to obtain very high yields of my protein. What kind of systems do you offer?
For stable high-yield expression, we offer the Freedom CHO-S and Freedom DG44 kits. For transient high-yield expression, we offer the ExpiCHO Expression System, Expi293 Expression System, FreeStyle 293 Expression System, FreeStyle Max 293 Expression System, and FreeStyle Max CHO Expression System. 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.
Do I need a license to use the Expi293 MembranePro Expression System?
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."
Do I need to remove Anti-Clumping Agent and Pluronic F-68 from the medium while growing Expi293 cells? How about during transfection of these cells?
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.
