The substrate specificity of CLIP-tag, SsOGT-H5 and SsOGT-MC8 proteins were evaluated on BG-N3 and BC-N3 substrates by the competitive inhibition assay performed as described [15] , [27] , [29] (link). By using fixed concentrations of the fluorescent BC-TMR substrate (5 μM) and enzymes (5 μM), an increasing concentration of guanine/cytosine-azide derivatives (0–1 mM) was added to the mixtures. The reactions were incubated for 60 min at 25 °C for CLIP-tag and 65 °C for MC8 respectively and then stopped by adding Laemmli buffer 1 ×. Subsequently, the samples were loaded on SDS-PAGE and the fluorescent bands were measured by gel-imaging on a VersaDoc 4000™ system (Bio-Rad), by applying green LED/605 bandpass filter. Then, obtained data were plotted by IC50 equation [15] , [25] . As control, 5 μM of SsOGT-H5 protein was incubated at 65 °C with 5 μM of the fluorescent substrate BG-FL in presence of an increasing amount of the BG/BC-azide derivatives (0–1 mM). In this case, fluorescent bands were visualized on VersaDoc 4000™ system (Bio-Rad), by applying a blue LED/530 bandpass filter.
Versadoc 4000 system
Manufactured by Bio-Rad
Sourced in United States
The VersaDoc 4000™ system is a compact and versatile imaging system designed for capturing high-quality images of gels, blots, and other samples. It utilizes a scientific-grade CCD camera and a range of illumination options to provide accurate and reproducible results. The system is capable of detecting a variety of imaging signals, including chemiluminescence, fluorescence, and visible light.
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4 protocols using versadoc 4000 system
1
Competitive Inhibition Assay for CLIP-tag, SsOGT-H5, and SsOGT-MC8 Substrate Specificity
2
Fluorescent Enzyme Activity Assay
3
In vitro analysis of S. solfataricus OGT activity
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The activity of in vitro-expressed SsOGT was analysed incubating 8 μg of pBS-rRNAp-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.).
4
Positive Supercoiling Assay with TopR1
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Positive supercoiling assay was performed using plasmid pBluescript (Qiagen) as a substrate, as previously reported [20 (link),33 (link)]. Briefly, different amounts of protein extracts (1–5 μg) prepared from E. coli H5-TopR1-expressing cultures or purified TopR1 (60 nM) were incubated, at 85°C for 10 min. in a total volume of 20 μL, with 300 ng of the substrate in 1× RG buffer (35 mM Tris-HCl, pH 7.0, 0.1 mM Na2EDTA, 30 mM MgCl2, 2.0 mM DTT, 1 mM ATP). Plasmid topoisomers were separated by 2D agarose gel electrophoresis with ethidium bromide (0.01 μg/ml) in the second dimension. After electrophoresis, gel was stained with ethidium bromide (1 μg ml-1), and analysed under UV light with a VersaDoc 4000™ system (Bio-Rad, Hercules, CA, USA).
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