Sypro ruby protein gel stain

Proteins (typically 20 μl per sample) were separated by SDS-PAGE NuPAGE 4–12% Bis-Tris Gels in 1X NuPAGE MOPS SDS (Life Technologies) and transferred into Immobilin P PVDF membranes (Millipore) in a tris-glycine buffer containing 10% methanol. Membranes were washed 3x5mins in PBS supplemented with 0.05% TWEEN20 (NBS Biologicals) (PBST) under gentle agitation, blocked in PBST supplemented with 5% w/v dried skimmed milk (Marvel) for 45mins and washed 3x5mins in PBST prior to incubation with antibody in PBST supplemented with 2.5% (w/v) milk or PBS at 4°C. Dilutions and antibodies used are specified in S2 Table. Proteins were visualised by ECL Western Blotting Detection Reagents (GE Healthcare), as per the manufacturer’s instructions. Molecular size was estimated from the mobility of Novex Sharp Pre-stained Protein Standards.
For protein identification 35 μl of per sample were separated, gels were fixed and stained using the SilverQuest Staining Kit (Life Technologies), or SYPRO Ruby Protein Gel Stain (Life Technologies), according to the manufacturer’s instructions. Bands of interest were excised and identified by tandem LC-MS/MS mass spectrometry, performed by the mass spectrometry facility at the MRC Laboratory of Molecular Biology, UK.

Tissue proteins were extracted from 40 HGSOC. Protein oxidation was measured by OxiProteomics (https://www.oxiproteomics.fr/). Extracted proteins were quantified by the Bradford method and split into equal amounts for analyses. Carbonylated proteins were labeled with specific functionalized fluorescent probes and samples were resolved by high-resolution electrophoresis separation. Total proteins were post-stained with SyproRuby protein gel stain (Life Technologies, #S12000). Image acquisition for carbonylated and total proteins was performed using the Ettan DIGE imager (GE Healthcare). Image processing and analysis was performed using ImageJ (Rasband, W.S., ImageJ, National Institutes of Health, USA, https://imagej.nih.gov/ij/, 1997-B014). Density histograms and lane profile plots were obtained from each sample, both for carbonylated and total proteins. Carbonylated protein signal was normalized by total protein signal for each sample in order to obtain the carbonylation score (Score = carbonylated protein / total protein).

C57BL/6 mice (6–8 weeks of age) were injected i.v. with histones (50 mg kg⁻¹) in normal saline. Retro-orbital bleeds were performed with non-heparinized tubes at 1–2 min post injection and collected in ACD. Isolated plasma from each mouse was then diluted 1/10 in PBS, 200 µl of the diluted plasma added to 100 µl of packed heparin-coupled Sepharose beads (Bio-Strategy) prewashed in PBS in a 0.5 ml Eppendorf tube and the mixture incubated for 30 min at 4 °C on a rotator. The beads were then pelleted by brief centrifugation (~10 s), the supernatant plasma collected, the beads washed twice with PBS, and proteins bound to the beads eluted by boiling in 100 µl of reducing SDS-PAGE sample buffer for 5 min. Eluted proteins were then run on 4–20% tris-glycine gels (Novex), and stained with SYPRO Ruby protein gel stain (Life Technologies). SYPRO Ruby images were captured using the Bio-Rad ChemiDoc Imaging System and Image lab software. ImageJ analysis was used to determine densitometry of histone bands and histone concentrations were determined with reference to histone standards. A 25 kDa unidentified, heparin-binding, protein present in mouse plasma was used as a loading control. When determining the effect of mCBS and MTS on circulating histone levels, experiments were performed as above, except 10 min prior to histone injection mCBS and MTS were injected i.p. at 100 mg kg⁻¹.