Expi293™ Expression Medium, 1 L - FAQs

View additional product information for Expi293™ Expression Medium - FAQs (A1435102, A1435101, A1435104, A1435103)

15 product FAQs found

Can I purify my protein from Expi293 Expression Medium using ProBond or Ni-NTA purification systems?

Expi293 Expression Medium is not directly compatible with ProBond or Ni-NTA purification systems. We recommend performing a buffer exchange or dialyzing the samples before His-tag purification.

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Which vector do you recommend using with the Expi293 Expression System?

We recommend using the pcDNA 3.4-TOPO TA vector (Cat. No. A14697). This vector contains the native, full-length CMV promoter and a WPRE (Woodchuck Posttranscriptional Regulatory Element) downstream of the cloning site, both of which contribute to high level gene expression (about 2-3 fold higher expression than with pcDNA3.3 TOPO vector, which in turn provides 2-5 fold higher expression than with a standard pcDNA vector). Of course, the expression level is also protein-dependent.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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 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.

Can I shake Expi293 cultures faster or slower than recommended?

For most flasks, there is a dropoff in expression when you go too fast or too slow because of cell shear stress or insufficient aeration. The effect is greater in plates where there is a sharper transition between static and moving fluid in the wells.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Do I need to change media after transfection of Expi293 cells?

No. The Expi293 Expression System is designed to run without media exchanges. There is no need to remove transfection complexes or to change growth medium following transfection.

With the Expi293 Expression System, for how many days should I wait before harvesting my protein?

The optimal expression time will be different for each protein, but most tested proteins fall within a 3-7 day window. Since the system scales well, it is recommended that you run a small-scale pilot experiment to determine when to harvest your protein of interest prior to scaling up.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

If I let my Expi293 cells grow to a higher density than recommended, will they be okay?

While Expi293 medium can support much higher cell densities, we do not recommend growing your Expi293 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 the cells 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 your own expression construct of known yield to determine if cell expression performance has been impacted.

My Expi293 Expression Medium (Cat. No. A1435102) was accidentally frozen. Can I still use it?

This product should be stored at 2-8 degrees C and should not be frozen. The biggest issue with accidentally freezing this product is its solubility. If this product was accidentally frozen, we recommend placing it in a 2-8 degree environment and allowing it to slowly thaw overnight. If the product is fully thawed and shows no signs of precipitation, then it should still be usable, but we cannot guarantee effectiveness.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Does Expi293 Expression Medium contain antibiotics?

Expi293 Expression Medium does not contain antibiotics. We do not recommend the use of antibiotics, however, 5 mL/L of Antibiotic-Antimycotic (Cat. No. 15240) containing penicillin, streptomycin, and amphotericin B may be used when required, as stated on page 1 in the manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/expi293_exp_medium_man.pdf).

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

Do I need to change media after transfection in the Expi293 Expression System?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I only added one enhancer and not the other with the Expi293 system. Will I still get good protein expression?

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.

What method do you recommend for quantifying the rabbit Antibody-Expressing Positive Control Vector (Cat. No. A14662)?

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.