Alexafluor 680 conjugated goat anti mouse igg secondary antibody

Equivalent amounts of protein were suspended in protein sample buffer (50 mM Tris pH6.8, 5% SDS, 10% glycerol, 5% β-mercaptoethanol, 25mM NaF, 1mM Na₃VO₄, 5mM β-glycerophosphate, 1mM PMSF, 50 μM Leupeptin, 100 μM Pepstatin A), resolved by electrophoresis in Bis-Tris polyacrylamide gels and transferred to polyvinylidene fluoride membranes by electroblotting. Membranes were blocked in TNET (10 mM Tris pH 7.5, 50 mM NaCl, 2.5 mM EDTA pH 8.0, 0.1% tween) supplemented with 5% nonfat dry milk. Primary antibodies used in these experiments were against: HA (#12013819001, Roche), FLAG (F3165, Sigma), SMCX (NB100-55328, Novus Biologicals), EP400 (A300-541A, Bethyl Laboratories), Brd4 (Schweiger et al., 2006 (link)), TIP60 (sc-16623, Santa Cruz Biotechnology) and actin (MAB1501, Millipore). Bound antibodies were detected with horseradish peroxidase conjugated anti-rabbit or anti-mouse secondary antibodies. For detection of actin and GAPDH, membranes were incubated with an AlexaFluor 680 conjugated goat anti-mouse IgG secondary antibody (Molecular Probes/Invitrogen) and labeled proteins visualized using a LI-COR Odyssey Infrared Imaging System.