Immobiline drystrips ph 4 7

To visualize the phosphorylation pattern of XRCC1, 200 μg of protein lysates from kinase inhibited HeLa S3 cells were separated by two-dimensional polyacrylamine gel electrophoresis (2D-PAGE) and visualized by western blot. 40 ng of recombinant Interferon regulatory factor 3 (IRF-3) (pI 5.17, MW 47.2 kDa) was added to each sample as an internal localization standard to monitor differential XRCC1 migration. 2D-PAGE was performed using Immobiline DryStrips pH 4–7 (GE Healthcare) and pre-cast 4–12% denaturing NuPAGE gels (Invitrogen). Western blot analysis was performed using mouse monoclonal XRCC1 antibody (ab1838, Abcam) and rabbit polyclonal IRF3 antibody (4962, Cell Signalling) as primary antibodies, followed by HRP conjugated secondary rabbit anti-mouse and swine anti-rabbit antibodies (Dako Denmark). Blots were developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and scanned in IS4000R Kodak imager (Fisher Scientific). Quantification of spot intensities was performed by using the Kodak Molecular Imaging software version 4.0.1. After subtracting background intensity values, ratios of densities of “head” and “tail” regions versus total XRCC1 intensity (head + tail) were calculated.

Two-dimensional electrophoresis (2-DE) was performed as previously described (Chen et al., 2014 (link); Chen et al., 2008 (link)). Unfractunated whole serum samples (10 μl) were lysed, rehydrated in lysis buffer (2 M thiourea, 8 M urea, 4% CHAPS, 1% dithreitol and 2% pharmalyte), and subjected to isoelectric focusing in 11-cm rehydrated precast Immobiline Drystrips pH 4–7 (GE Healthcare Bioscience, Uppsala, Sweden) overnight, using the Protean Isoelectric Focusing Cell (Biorad Inc., Berkeley, CA, USA). The second-dimensional separation of focused samples in the gel strips was performed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% linear polyacrylamide gels. A previously described silver staining method was used to develop the 2-DE gels (Heukeshoven & Dernick, 1988 (link)), and a modified silver staining technique was used for mass spectrometry (MS) analysis (Snevechenko et al., 1996 (link)). All samples were analysed in duplicate.