Irdye680 conjugated anti rabbit igg

Crude protein extracts were prepared using a Trichloroacetic Acid (TCA) precipitation as described by (Peter et al., 1993 (link)). Briefly 5 ml of cells were harvested by centrifugation for 2 min at 3500 rpm. Cell pellets were resuspended in 0.5 ml of ice-cold buffer A (20 mM Tris (pH 8.0), 50 mM NH₄OAc, 0.5 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride) and were immediately mixed with 0.5 ml of ice cold 20% trichloroacetic Acid. Cells were then vortexed in the presence of glass beads for 2 × 0.5 min, with 2 min cooling on ice in-between. The supernatant was the placed into a new tube, proteins pelleted by centrifugation for 10 min at 12,000 rpm, and the pellet resuspended in trichloroacetic acid-sample buffer (3% SDS, 100 mM Tris base pH 11, 3 mM DTT). Extracts were then boiled for 10 min and insoluble cell debris pelleted by centrifugation for 2 min at 12,000 rpm. Proteins were separated by SDS-PAGE polyacrylamide gene electrophoresis followed by immunoblotting to a nitrocellulose membrane. Immunoblots were probed with anti-GFP (G1544, Sigma) and anti-Act1 (ab3280–500) primary antibodies and IRDye800CW-conjugated anti-mouse IgG (LI-COR) and IRDye680-conjugated anti-rabbit IgG (LI-COR) secondary antibodies. An Odyssey infrared image system (LI-COR) was used for visualization and analysis of signal intensities.