Samples were either run on 4–12% NuPAGE or Bolt gels with the NuPAGE Bis–Tris system (Invitrogen) in MOPS SDS buffer or on 8% gels with the Bio‐Rad system with SDS‐running buffer. Western blotting was performed with the wet transfer systems from Bio‐Rad on 0.45 μm Nitrocellulose membranes (Amersham™), which were blocked in 5% milk in PBS. Primary antibody dilutions were 1:500 in 5% milk‐PBS, while secondary antibodies were diluted 1:2,000. Blots were visualized with SuperSignal™ West Pico PLUS or Femto Chemiluminescent Substrate solution (Thermo Fischer) or with Clarity™ Western ECL solution (Bio‐Rad) and scanned on Fusion FX7 imaging system (Witec AG).
Nupage bis tris system
Manufactured by Thermo Fisher Scientific
The NuPAGE Bis–Tris system is a polyacrylamide gel electrophoresis (PAGE) system designed for the separation and analysis of proteins. It utilizes a Bis-Tris buffer system and pre-cast gels to provide consistent and reliable protein separation results.
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2 protocols using nupage bis tris system
1
Western Blot Sample Preparation and Imaging
2
RNase-Assisted RNA Interactome Profiling
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RNase-assisted RNA chromatography with RNAse A/T1 was performed, using in vitro-transcribed pri-mir-30c-1 and total MCF7 cell extracts28 (link). RNA-bound proteins were separated using 4–12% NUPAGE bis–tris system (Invitrogen) and individual lanes were subjected to mass spectroscopy (BSRC Mass Spectrometry and Proteomics Facility, University of St Andrews). Results were confirmed by western blot analysis with anti-SRSF3 (MBL), anti-PARP-1 (Santa Cruz), anti-DDX17 (Santa Cruz), anti-hnRNPA1 (Thermo Scientific) and anti-FUS (Abcam) specific antibodies, as indicated above (Western blot analysis section).
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