Ecl plex goat anti rabbit igg cy5 secondary antibody

Cells were grown to near confluence, collected, and fractionated into subcellular components (Subcellular Protein Fractionation Kit for Cells, ThermoScientific, Rockford, IL, 61101). The subcellular fractions were run on a precast polyacrylamide gel (BioRad, Hercules, CA, 94547), along with Full-Range Molecular Weight Rainbow Markers (GE Healthcare Life Sciences, Piscataway, NJ, 08855) then transferred to a PVDF membrane. Western analysis was performed on the membrane with a rabbit polyclonal antibody against human nucleolin (Abcam, Cambridge, MA, 02139), and ECL Plex goat anti-rabbit IgG Cy5 secondary antibody (GE Healthcare Lifesciences, Piscataway, NJ, 08855). The blot was analyzed on a Typhoon 9410 variable mode imager (GE Healthcare Lifesciences, Piscataway, NJ, 08855) and quantified.

Wildtype and mutant SMYD3 cDNAs were transferred from Gateway pENTR into pEF-DEST51 (N-terminal V5-tagged) by TOPO cloning. NIH3T3 cells were transiently transfected, harvested 48 hours later, and then lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 50 mM Tris pH 8, 0.1% SDS) containing protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN). Expression levels were determined by Western blotting. Proteins were resolved on 8–15% SDS-PAGE, transferred to nitrocellulose (Protran BA, Schleicher and Schuell, NH), and blocked using 5% nonfat milk (10g nonfat milk, 150 mM NaCl, 10 mM Tris pH 8, 0.05% Tween-20) overnight at 4°C. Membranes were incubated with anti-SMYD3 polyclonal antibody [19 (link)] for 1 hour at room temperature, extensively washed, then incubated with ECL Plex Goat anti-Rabbit IgG-Cy5 Secondary Antibody (GE Healthcare) for 1 hour at room temperature. Blots were exposed and developed using ECL blot detection reagent (Amersham Pharmacia Biotech) according to manufacturer's instructions.