Ecl plex goat α mouse igg cy5

Protein nuclear extracts were obtained as described in the pull-down experiment. Biotinylated oligonucleotides 150 pmol, folded in phosphate buffer pH 7.4 100 mM and KCl 100 mM, were bound to 50 μl of streptavidin-coated magnetic beads. DNA coupled-beads were incubated with nuclear proteins 150 μg extract at 4°C for 90 min and proteins excess was washed with Tris-HCl pH 7.5 50 mM-NaCl 150 mM solution. Samples were fixed with formaldehyde (Sigma-Aldrich, Milan, Italy) 5% for 30 min at room temperature, washed and then analyzed by western blot analysis, with an anti-hnRNP K antibody (mouse monoclonal D-6; Santa Cruz Biotechnology, Dallas, TX, USA). Briefly, samples were electrophoresed on a 10% SDS-PAGE and transferred to a nitrocellulose blotting membrane (Amersham TM Protan TM, GE Healtcare Life science, Milan, Italy) by using trans-blot SD semi-dry transfer cell (Bio-Rad Laboratories, Milan, Italy). The membrane was blocked with 2.5% skim milk in phosphate-buffered saline solution, incubated with the anti-hnRNP K antibody and then with the ECL Plex Goat-α-Mouse IgG-Cy5 (GE Healthcare Life sciences, Milan, Italy). At last, images were captured on Typhoon FLA 9000.

Immunoblot analysis was performed on cell protein extracts obtained in RIPA Buffer (20 mM Tris-HCl pH 7.5; 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 1 mM Na₃VO₄, 1x protease inhibitors). Protein concentrations were quantified using the Pierce^® BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Each sample was electrophoresed on 10% SDS-PAGE and transferred to a nitrocellulose blotting membrane (Amersham TM Protan TM, GE Healtcare Life science, Milan, Italy) by using trans-blot SD semi-dry transfer cell (Bio-Rad Laboratories, Milan, Italy). The membranes were blocked with 2.5% skim milk in PBST (0.05% Tween 20 in PBS). Membranes were incubated with the respective primary antibody directed against hnRNP A2/B1 (mouse monoclonal; Santa Cruz Biotechnology, Dallas, TX, USA), alpha-tubulin (mouse monoclonal; Sigma-Aldrich, Milan, Italy). After three washes in PBST, membranes were incubated with ECL Plex Goat-α-Mouse IgG-Cy5 (GE Healthcare Life sciences, Milan, Italy). Images were captured on the Typhoon FLA 9000.