Amersham biosciences ecl prime western blotting detection reagent

Competence pili preparations were obtained from S. pneumoniae grown in 500 ml of C+Y medium, and competence was induced as described above. Bacteria were pelleted at 4 °C by centrifugation for 15 min at 6000 × g. The supernatant containing detached/broken pili was filtered and pelleted by ultracentrifugation at 100,000 × g at 4 °C for 1 h. Pellets were resuspended in 100 μl of PBS. Multimerization of mature ComGC was assessed by two-dimensional PAGE, native gel (first dimension) and SDS-PAGE (second dimension). In brief, pili preparations of competent T4 WT and T4ΔcomGC were run on a 12% native gel. Then, one entire lane of each sample was cut and placed perpendicular on top of a second gel. After migration, electroblotting (Bio-Rad, Trans-Blot® Turbo^TM Midi PVDF Transfer Packs) and immunodetection with ComGC antibody were performed. Rabbit polyclonal ComGC antibody has been previously described (37 (link)). HRP-conjugated goat anti-rabbit antibody (GE Healthcare) and Amersham Biosciences ECL Prime Western blotting detection reagent (GE Healthcare) were used to visualize the blots.

Cell line pellets were washed in PBS and then incubated with lysis buffer (1% Triton X-100, 150 mm NaCl, 10 mm Tris (pH 7.4), 1 mm EDTA, 1 mm EGTA (pH 8.0); 0.2 mm Na₃VO₄, and 0.2 mm NP-40). 100 μg of protein lysates were resolved using SDS-PAGE electrophoresis and then transferred to nitrocellulose membranes. The anti-p53 antibodies DO-1, DO-7, and 1801 (Santa Cruz Biotechnology, Dallas, TX) were used at 0.8 μg/ml and incubated overnight. Because Δ40p53 lacks the 40 N-terminal amino acids (Fig. 1, region TAD 1), it can be distinguished from fl-p53 using a combination of the anti-p53 monoclonal antibodies DO-1 or DO-7 and 1801, which bind to this region. The ECL detection reagent (Amersham Biosciences ECL Prime Western blotting detection reagent, GE Healthcare) was used to detect antigen–antibody complexes. Membranes were imaged using the ChemiDoc MP imaging system (Bio-Rad).