Immunoprecipitation of FT2Fc from Cell Cultures

Immunoprecipitation (IP) of FT₂Fc from cell culture supernatant was performed as previously described with minor modifications [23 (link)]. Briefly, sheep-anti mouse IgG paramagnetic beads (Dynal, 11201D) were coupled with anti-His mAb (Invitrogen, 37–2900) in coating buffer (PBS plus 0.1% immunoglobulin-free BSA) for 2 h at RT on a rotating wheel. Three molar excess of His-tagged Fab83 were added. After 1 h incubation, beads were washed three times with coating buffer. Cell culture supernatant was diluted 1:1 in IP buffer (50 mM Tris-Cl, 75 nM NaCl, 1% Igepal, protease inhibitor mixture (Sigma, 11836153001), pH 7.4) plus 0.5% BSA and incubated with 50 μl of Fab83-anti-His antibody coupled beads. The same input was used for all conditions. IP was performed overnight at 4°C. After five washes with 50 mM Tris-Cl, 0.5% Igepal, 150 nM NaCl, 0.5% BSA, pH 7.4, elution of immunoprecipitated FT₂Fc was performed by incubation with peptides (200 molar excess compared to Fab83) for 2 h at 4°C. The eluate was boiled in 4 x LDS for western blotting. After elution, the beads were boiled in 4 x LDS and the supernatant was investigated by western blotting.