Versadoc apparatus

The activity of in vitro-expressed SsOGT was analysed incubating 8 μg of pBS-rRNA_p-ogt plasmid or 200 ng of recombinant SsOGT OGT with 200 μg of S. solfataricus whole cell extract under the experimental conditions described above for coupled in vitro transcription/translation and in the presence of BG-FL substrate (2.5 μM). The mix reaction was incubated at 70°C for 60 min. Reactions were stopped by denaturation, and samples were subjected to SDS-PAGE, followed by fluorescence imaging analysis using a VersaDoc 4000™ system (Bio-Rad Laboratories Inc.) by applying as excitation/emission parameters a blue LED bandpass filter. For western blot analysis, proteins were transferred onto PVDF filters (Bio-Rad Laboratories Inc.) using the Trans-Blot® Turbo™ Blotting System (Bio-Rad Laboratories Inc.). The presence of SsOGT protein was revealed using polyclonal antibodies raised in rabbit against S. solfataricus OGT as primary antibodies, the goat anti-rabbit IgG-HRP (Pierce) as secondary antibody, and the Amersham Biosciences ECL Plus kit. Filters were incubated, washed, and developed according to the manufacturer's instructions. Chemiluminescent bands were revealed using a VersaDoc apparatus (Bio-Rad Laboratories Inc.).

Total and fractionated protein extracts were prepared as previously described [22 (link)]. Samples were run in 4–12% gradient gels in 1x Tris-Glycine SDS Running buffer (25 mM Tris, 192 mM glycine and 0.1% SDS, pH 8.3). After electrophoresis, proteins were transferred onto PVDF filters (Bio-Rad) using the Trans-Blot^® Turbo^™ Blotting System (Bio-Rad). Reagents used were: polyclonal antibodies raised in rabbit against S. solfataricus OGT [9 (link)] and TopR1 reverse gyrase [21 (link)] as primary antibodies; the goat anti-rabbit IgG-HRP (Pierce) as secondary antibody and the Amersham Biosciences ECL Plus kit. Filters were incubated, washed and developed according to manufacturer’s instructions. Chemiluminescent bands were revealed using a VersaDoc apparatus (Bio-Rad) and the QuantityOne software (Bio-Rad) was used for quantitation.

Protein extracts were prepared as described above and incubated for 5 min. at 100 °C. Different amounts of proteins were loaded on 4–20% gradient acrylamide gel and transferred onto PVDF membrane. After incubation with corresponding antibodies, the protein analysis was performed using ECL-PLUS kit (GE-Healtcare) as described before and VersaDoc apparatus (Bio-Rad) as previously described^{35 (link)}. Primary antibodies used: Anti-FLAG -HRP conjugated antibody (abcam; 1:10000), Anti Actin (abcam 1:500) and Anti RAD-51 (1:1000).