Samples were prepared as previously described [28 (link)], resolved on Bolt pre-cast 4-12% gels (Thermo Fisher Scientific), using Bolt MES SDS running buffer (Thermo Fisher Scientific) and transferred onto nitrocellulose membranes. After a blocking step in 5% non fat-dried milk in phosphate-buffered saline (PBS)-0.1% Tween, membranes were incubated with primary antibodies overnight at 4 °C. After three washes in PBS - 0.1% Tween, membranes were incubated with the appropriate HRP-linked secondary antibodies (Bio-Rad Laboratories) for 45 min at room temperature, washed with PBS-0.1% Tween and analysed by chemi-luminescence (GE Healthcare Life Science). Images were acquired and quantified using Alliance Mini HD6 system by UVITEC Ltd., Cambridge, equipped with UVI1D Software (UVITEC, 14-630,275). Detailed information for all antibodies is provided in the Supplementary Table S1 .
Alliance mini hd6 system
Manufactured by Uvitec
The Alliance Mini HD6 system is a compact and versatile laboratory equipment designed for various applications. It features high-definition video capture and image processing capabilities. The system provides users with essential functionalities for their research and analytical needs.
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5 protocols using alliance mini hd6 system
1
Western Blot Analysis of Protein Expression
2
R-loop Detection by Slot Blot
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Genomic DNA was extracted according to DRIP protocol. Serial dilutions of DNA were double spotted on a nitrocellulose membrane and crosslinked with UV light (700 mJ/cm2). One part of the membrane was blocked with TBS‐Tween 0.1 and 5% nonfat dry milk (NFDM) for 1 h and then incubated with S9.6 antibody diluted to 0.5 μg/ml in TBS‐Tween plus 2% NFDM. After washing, membrane was incubated with anti‐mouse secondary antibodies, further washed, and developed with ECL techniques. The other part of membrane was incubated with Methylene Blue (Sigma M4159 0.2% (w/v) in 0.4 M sodium acetate:0.4 M acetic acid) for 15 min, as loading control, and at the end of the incubation washed with water. Both the membranes were acquired and quantified using Alliance Mini HD6 system by UVITEC Ltd, Cambridge, equipped with UVI1D Software (UVITEC, 14–630,275).
3
Western Blot Quantification Protocol
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Samples were prepared as previously described (27) , resolved on Bolt pre-cast 4-12% gels (Thermo Fisher Scienti c), run using Bolt MES SDS running buffer (Thermo Fisher Scienti c) and transferred onto nitrocellulose membranes. After a blocking step in 5% non fat-dried milk in phosphate-buffered saline (PBS)-0.1% Tween, membranes were incubated with primary antibodies overnight at 4°C. After three washes in PBS - 0.1% Tween, membranes were incubated with the appropriate HRP-linked secondary antibodies (Bio-Rad Laboratories) for 45 min at room temperature, washed with PBS-0.1% Tween and analysed by chemi-luminescence (GE Healthcare Life Science). Images were acquired and quanti ed using Alliance Mini HD6 system by UVITEC Ltd, Cambridge, equipped with UVI1D Software (UVITEC, 14-630275). Detailed information for all antibodies is provided in the Supplementary Table S1.
4
Western Blot Protocol for Protein Detection
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Samples were separated by electrophoresis and transferred onto nitrocellulose membranes. After a blocking step in 5% nonfat-dried milk in 0.1% Tween-PBS, membranes were incubated with primary antibodies overnight at 4°C. After three washes in 0.1% Tween-PBS, membranes were incubated with the appropriate HRP-linked secondary antibodies (Bio-Rad) at room temperature for 45 min, washed with 0.1% Tween-PBS and analysed by chemi-luminescence (GE Healthcare Life Science). Images were acquired and quantified using Alliance Mini HD6 system by UVITEC Ltd, Cambridge, equipped with UVI1D Software (UVITEC, 14–630275). Detailed information for all antibodies is provided in Supplementary Table S2 .
5
Western Blot Analysis of Che-1 and β-Actin
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Cells were treated as described in Bruno T. et al., 2006 (44 (link)). Samples were separated by electrophoresis and transferred onto nitrocellulose membranes. After a blocking step in 5% non-fat-dried milk in 0.1% Tween-PBS, membranes were incubated with primary antibodies overnight at 4°C. After three washes in 0.1% Tween-PBS, membranes were incubated with the appropriate HRP-linked secondary antibodies (Bio-Rad, IT) at room temperature for 45 min, washed with 0.1% Tween-PBS and analyzed by chemi-luminescence (GE Healthcare Life Science, IT). Images were acquired using Alliance Mini HD6 system by UVITEC Ltd, Cambridge, equipped with UVI1D Software (UVITEC, 14–630275). The primary antibodies used were: anti-Che-1 (43 (link)), and anti-β-actin (Sigma – Aldrich, Merck, IT).
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