Magna bind goat anti mouse igg magnetic bead slurry

A total of 400 μL of plasma was used from each group to immunoprecipitate Ago2 protein in order to isolate Ago2 coupled miRNAs. Each plasma sample was diluted with 400 μL of DPBS (pH 7.4) resulting 800 μL of diluted plasma. Following this, 400 μL of Magna Bind goat anti-mouse IgG Magnetic Bead slurry (Thermo Scientific, Rockford, USA) were washed in DPBS and incubated with 10 μg of mouse monoclonal anti-Ago2 mouse normal IgG (Santa Cruz Biotechnology Inc, USA) antibodies for 2 h at 4 °C. The pre-incubated beads and antibody were then added to the 800 μL of diluted plasma and incubated overnight at 4 °C. Beads were washed three times with 1 % Nonidet P-40 buffer (1 % Nonidet P-40, 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA (BD Bioscience, Heidelberg, Germany). After the final wash 800 μL of QIAzol lyses buffer was added directly to the Ago 2 pellet and processed for RNA isolation.

Magna Bind goat anti-mouse IgG magnetic bead slurry, 100 μL, (Thermo Scientific, Waltham, USA) was incubated with 10 μg of mouse monoclonal anti-Ago2 (Abcam, Cambridge, UK) or mouse normal IgG (Santa Cruz Biotechnology, Dallas, US) antibodies for 2 h at 4 °C. The antibody-coated beads were then added to plasma and incubated overnight at 4 °C with rotation. Beads were washed and each sample then eluted in RNAse free water before QIAzol was added for RNA isolation. Ago2 isolation was determined by western blot analysis.

An AGO2-specific antibody was used for AGO2 immunoprecipitation, and an IgG antibody was selected as the negative control. Mouse monoclonal anti-AGO2 (Abcam, Cambridge, England) or mouse normal IgG antibody (Abcam, Cambridge, England) were preincubated with Magna Bind goat anti-mouse IgG Magnetic Bead slurry (Thermo Fisher Scientific, Waltham, MA, United States) and used for immunoprecipitation. In brief, cells were lysed in 150 mM KCl, 25 mM Tris-HCl, pH 7.4, 5 mM EDTA, 0.5% Triton X-100, and 5 mM DTT supplemented with RNase inhibitor (Takara, Tokyo, Japan) and proteinase inhibitor cocktail (Roche Applied Science, Basel, Switzerland). The lysate was mixed with antibody-coupled Sepharose beads and left under rotation for 4 h at 4°C. Beads were subsequently washed six times in lysis buffer and the RNA was extracted using Trizol reagent(Takara, Tokyo, Japan).