Fluorescent dye labelled secondary antibody

The ubiquitination experiments were conducted as follows: E1 (60 nM), Ubc4p (200 nM), Ufd4p (300 nM), Ufd2p (200 nM) and Ub-V-GFP (400 nM) were incubated with FLAG-Ub (10 nM) at 30 °C in buffer containing 25 mM Tris–HCl (pH 7.4), 2 mM magnesium/ATP and 0.1 mM DTT. Ubiquitination of Ub-V-GFP was detected by immunoblotting with an anti-GFP antibody. The immunoblotting was performed using a fluorescent dye-labelled secondary antibody (Invitrogen, Carlsbad, CA) and the blots were scanned using an Odyssey infrared imager.

Heart extracts were prepared in cold RIPA-like buffer (25 mM Tris HCl, pH 7.6, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl fluoride and a protein inhibitor cocktail; 04693132001, Roche Diagnostics, Basel, Switzerland) for 30 min on ice after sonication. The homogenates were centrifuged at 12 000 r.p.m. for 15 min at 4°, and the protein concentrations were determined by the Bio-Rad protein assay. The protein lysates (approx. 25 µg) were electrophoresced under reducing conditions in SDS-PAGE gels and transferred onto nitrocellulose membranes. After incubating in primary antibody, immunoblotting was performed using a fluorescent dye-labelled secondary antibody (Invitrogen), and the blots were scanned using an Odyssey infrared imager.