Antibody-Expressing Positive Control Vector

The above vector has coding sequences for expression of the rabbit IgG1 antibody.

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The optimal complexation time is 5 minutes. We have observed a small drop in protein yield (approximately 20% reduction) if complexes sit up to 10 minutes; for 20 minutes or longer yield will be drastically reduced (>50% reduction in yield).

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Yield can vary greatly from protein to protein. We strongly recommend expressing the rabbit antibody positive control (Cat. No. A14662) to determine if the low yield is due to a low expressing protein, a problem with the system components, or transfection and culturing conditions. If you are not achieving the expected yield with the rabbit IgG positive control, we recommend checking the following:

- Ensure that cells are >95% viable during normal passaging and at time of transfection
- Have a doubling time of approximately 17 h
- Recover cells rapidly post-thaw (within 3-4 days post thaw); if not, verify you are using the culturing guidelines provided in the manual and thaw a new vial of cells if necessary.
- We recommend gentle swirling at all handling steps for ExpiCHO-S cells (avoid vigorous swirling, shaking, and pipetting up and down). Verify that your shake speed is about 110-125 rpm for 25 mm throw shakers and about 125 for 19 mm throw shakers. Test a flask with containing water and a thermometer to determine whether the shaker is putting off excess heat; reduce the incubator temperature setting as necessary to achieve a final temperature of 37 degrees C for your cultures. All of our shake speed recommendations are provided for Corning non-baffled flasks; if you are using a different culture vessel, additional shake speed optimization may be required.

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Some mild cell clumping is normal during ExpiCHO-S cell passaging and is okay as long as the cells have >95% viability. At each passage you may allow the clumps to settle to the bottom by letting the flask rest for 30 sec to 1 min, then passage the suspended cells. If the cells have a lot of clumping paired with viability < 95%, this indicates a problem either with the culture conditions or the cells themselves. We recommend verifying that the cells are: cultured according to the recommendations in the manual, below passage 20, and free of contamination. Thaw a new vial of cells as necessary.

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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.