Bio dot sf microfiltration apparatus

Cellular fractionation was carried out to isolate nuclei from HeLa cells (approximately 20×10⁶) collected 24 h post siRNA treatment (Ayala et al., 2006 (link)). Cell pellets were resuspended in 3 ml of buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM TCEP, EDTA-free protease inhibitor cocktail). The cell membrane was disrupted with 20 strokes of a pre-chilled Dounce homogenizer using a tight pestle. Nuclei were pelleted by centrifugation (1000 g for 5 min) and rinsed in buffer A. All steps were performed at 4°C. Total DNA was isolated from the nuclear pellet using QIAamp DNA mini kit (Qiagen) according to manufacturer's instructions. For slot blot analysis of S9.6 and double-strand (ds)DNA, DNA (0.25 and to 0.5 μg) was loaded on Hybond N+ (GE-Healthcare) using a Bio-Dot SF microfiltration apparatus (Bio-Rad) according to the manufacturer's instructions before crosslinking (120 mJ/cm²). Membranes were blocked in 5% blotting grade blocker (Bio-Rad) in TBST (Tris-buffered saline containing 0.1% Tween) and probed with S9.6 (1:1000; Kerafast ENH001) or dsDNA (1:2000; Abcam ab27156) in 2% blotting grade blocker in TBST overnight at 4°C. Blots were quantified by near-infrared western blot detection (LI-COR Biosciences). RNaseH treatment was carried out with 0.5 μg total DNA and 5 U RNase H1 (New England Biolabs) overnight at 37°C.

The 5′-end ³²P-labeled RNA substrates shown in Figure 3 (purchased from Dharmacon) were incubated with a serial dilution of cTSN (0 to 2 μM) in binding buffer containing 50 mM HEPES (pH 7.0), 250 mM NaCl, 5 mM β-Mercaptoethanol and 2 mM EDTA for 30 min at room temperature. The cTSN–RNA complex solutions were passed through a filter-binding assay device (Bio-Dot SF microfiltration apparatus, Bio-Rad). The isotope signals from cTSN–RNA complex bound on the nitrocellulose membrane and the free RNA passed through the filter-binding assay apparatus were exposed to a phosphorimaging plate and visualized by autoradiography (Fujifilm, FLA-5000). The intensities of cTSN–RNA complex and free RNA were quantified and analyzed by GraphPad Prism 4. Binding percentages from three measurements were calculated to derive the apparent K_d between cTSN and RNA by a one-site binding curve fitting using the equation Y = X/(K_d + X), where X is the cTSN concentration and Y is the percentage of bound RNA/total RNA.

SDS-PAGE was performed on 12% polyacrylamide gels using the Laemmli buffer system. Proteins were silver stained or transferred onto nitrocellulose membranes (Millipore) for Western blot analysis and probed with rabbit anti-saporin anti-serum 1:2000 or anti-His polyclonal antibody (Antibodies-online GmbH) and then with an anti-rabbit IRDye 680 secondary antibody 1:15.000 or an HRP-conjugated secondary antibody 1:20,000 followed by detection with ECL (Millipore). For quantitative determination, band intensity was measured by ImageJ software on images from silver-stained gels or non-saturated exposures of the films.
A Bio-Dot SF Microfiltration Apparatus (Bio-Rad) with Protran nitrocellulose membranes was used for slot blots, essentially following manufacturer’s instructions. Seed SAP was loaded as a reference standard in replicates.

Twelve micrograms of protein extracts (quantified used the BCA assay as described above) were prepared in a volume of 100 μL of RIPA buffer and loaded onto 0.2 μm cellulose acetate membrane (Whatman, GE Healthcare, United Kingdom) and filtered through a Bio-Dot SF Microfiltration Apparatus (BioRad, Italy). Slot-blots were probed as described for WB to detect retained SOD1 insoluble species. Densitometric optical analysis of slot-blots and their relative ponceau (used for loading control) was performed and represented as mean ± SEM.