Novex 6 tris glycine gel

For SDS-PAGE western blot the samples obtained in the Ago2 pull down assays were mixed with an appropriate amount of loading dye (25 mM Tris, 192 mM Glycine, 20% v/v glycerol, 4% m/v SDS, 0.1% v/v bromophenol blue in milli-Q water). The samples were denatured at 95°C for 5 minutes before electrophoresis on a Novex 6% Tris-glycine gel (Invitrogen). The samples were transferred to an Immobilin-P Transfer Membrane at 25V for 50 minutes. The membrane was then blocked for 60 minutes in 5% milk powder in PBS. The membrane was washed with 0.1% milk-PBS and incubated overnight at 4°C in 10 ml 0.1% milk-PBS with anti-FLAG primary antibody (1:5000) or alpha-actin (1:5000) with slow orbital mixing. After overnight incubation, the membrane was washed 3–4 times for 5 minutes with 0.1% milk-PBS and then incubated for at least one hour with slow orbital mixing in 10 ml 0.1% milk-PBS with goat anti-mouse secondary antibody (1:5000). The membrane was washed 2 times for 5 minutes in PBS-Tween, 2 times for 5 minutes in PBS and 2 times for 5 minutes in deionized H₂O. The Western Lightning ECL system (PerkinElmer Life Sciences) was used for luminometric detection with the LAS4000.