Mammalian Cell Expression
Mammalian Transient Expression Test
40ug of low endotoxin DNA (produced by mtibio) will be used to transfect 30mL of mammalian cells (either HEK293 or CHO) in suspension culture. A positive transfection control will be run in parallel using a beta-galactosidase expressing construct. At 2, 3, 4, 6, and 8 days post-transfection, 4ml culture samples will be taken, separated into cell and media fractions then flash frozen in liquid nitrogen and stored at -80C. All media and cell samples will be analyzed by SDS-PAGE.
Delivered: Cell and media samples; summary of expression results with images of SDS-gels.
Time: 2 weeks.
Mammalian Transient Expression - Affinity Analysis
Choose (A) or (B):
(A) His- or GST-tagged proteins: Lysis in buffer (standard lysis buffer is 50mM Tris-HCl, pH8.0; 150mM NaCl; 10% glycerol; 0.5mM TCEP; protease inhibitor cocktail), clarification by centrifugation, mixing with either Ni-NTA- or glutathione-agarose, recovery and washing of affinity resin. Elution (imidazole or reduced glutathione, respectively) and analysis by SDS-PAGE.
(B) Western Blot: Lysis in standard buffer (above), clarification by centrifugation followed by fractionation of clarified lysate by SDS-PAGE and blotting to nitrocellulose membrane. Blot will be blocked and treated with primary antibody supplied by customer (or anti-FLAG Ab) with washing (Tris-buffered saline with Triton X-100) and treatment with secondary antibody (goat anti mouse peroxidase conjugated) and staining using DAB (diaminobenzadine) as substrate.
Delivered: Summary of expression experiment including annotated images of stained SDS-gel or developed western blot.
Time: 1 week.
Mammalian Transient Expression Scale Up
Mammalian suspension cultures (HEK293 or CHO) will be expanded to the target volume (1L to 10L) and density (1x10e6 cells/mL) and transfected with endotoxin-free DNA (~1.2mg/L). The culture will be harvested at the optimal time as indicated in the expression analysis. Cell pellets will be flash frozen in liquid nitrogen and stored at -80C. If required, media will be processed according to customer preference for an extra charge depending on necessary material and protocol (please inquire).
Delivered: Weighed and frozen cell pellets.
Time: 2-3 weeks.