Protein g beads

Experiments were carried out as described previously (27 (link)). Cell lysates were prepared in modified RIPA lysis buffer and clarified by centrifugation. After being pre-cleared with protein G beads (GE Healthcare Life Sciences), the lysates were incubated with anti-GFP or anti-HA antibodies in cold room for 4 hr, followed by capture of the immunocomplexes with protein G beads for 2 hr. The beads were washed three times with lysis buffer to remove non-specifically bound proteins, and the immunoprecipitates were analysed by immunoblotting as described above.

For each immunoprecipitation, 30 µl of Protein-G beads (GE Healthcare) were prepared at 4°C by preincubating with 5 µg of anti-His antibody (GE Healthcare) for 30 minutes, blocking with 500 µl of 5% BSA containing 200 µg/ml sheared salmon sperm DNA for 30 minutes, and then washing twice with 250 µl of Wash Buffer (40 mM Tris pH 8, 4 mM MgCl₂, 70 mM KCl, and 7.5% glycerol).
Digested C. trachomatis serovar L2 434/Bu genomic DNA (10 pM) was incubated in the presence or absence of rEUO (20 nM) on ice for 30 minutes and then incubated with an aliquot of prepared Protein-G beads for a further 30 minutes. The supernatant was discarded and the beads were washed four times with 250 µl of Wash Buffer. Protein/DNA complexes were eluted from the beads with 100 µl of 0.1 M glycine pH 2.5, followed by addition of 12 µl of 1 M Tris pH 8.5 to neutralize the pH. Samples were heated to 95°C for 10 minutes to denature the protein. The DNA immunoprecipitation was performed as three independent experiments.