| Comments |
"After electrophoresis, [researchers] transferred all proteins to a blotting membrane. After equilibrating the gel in low SDS transfer buffer (39 mM glycine, 48 mM Tris base, 0.01% SDS, 20% methanol) for 15 min, [they] electrophoretically transferred proteins from the gel to
a thick, small-pore PVDF membrane (Immobilon PSQ. Millipore) using the Criterion or mini-Protean blotter (Bio-Rad) as directed by the manufacturer (100 V for 40–60 min). After transfer, [they] incubated the membrane for 1 h in 1% blocking reagent (Roche) in Tris-buffered saline (TBS) (150 mM NaCl, 10 mM Tris·HCl, pH 7.5) and then incubated overnight at 4 °C with primary antibodies and 1% blocking reagent in TBS with 0.05% Tween20. Table S1 contains a list of the sources and dilutions of all antibodies except for the anti-GFP antibody (JL-8 mouse antibody, 1:2,000 dilution Clontech)." |