Production of soluble CEACAM domains in human cells

Materials
Plasmid DNA
DMEM	Cell culture medium with L-glutamine and 4.5 g/l glucose (PAA Laboratories)
10% CS	calf serum (PAA Laboratories)
2xHBS	16.4 g NaCl, 11.9 g HEPES, 0.21 g Na2HPO4, solve in 1 l ddH2O, pH 7, sterile filtration
CaCl2 solution	2.5 M CaCl2
OptiMEM	Serum-reduced medium with L-glutamine and HEPES (Gibco)

Procedure

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HEK293T-cells are transfected with 5 µg plasmid DNA, coding for N-terminal domains of different receptors. After 6-8 hours the DMEM-medium is replaced by 5ml serum-reduced OptiMEM-medium. Due to the lack of a transmembrane domain, the N-terminal domains are secreted into the medium. After three days, the medium is collected in a 50 ml tube and centrifuged for 15 min at 5000 rpm to remove cell fragments. The supernatants containing the soluble proteins are stored at -20°C.