GST-GDI was then recovered onto glutathione-Sepharose beads, which were then resuspended in SDS gel sample buffer and Rab5 bound to GST-GDI was analyzed by SDS-PAGE and western blotting.
Iron metal was then recovered from the BOF slag.
The sulfate-sulfur was then recovered by precipitation as BaSO4.
The supernatant was then recovered into a new tube.
The acid-treated tRNA was then recovered by ethanol precipitation.
The affinity matrix was then recovered by centrifugation for 4 min at 16 000 g, and was then washed twice by resuspension in 0.2 ml of PBS buffer, and centrifugation for 4 min at 16 000 g through a 0.5 ml cushion of 10% sucrose in PBS buffer.
For leptomycin B (Sigma, L2913) treatment, ∼100 young adults were incubated for 2.5 hours at 25°C in 50 μl M9 buffer containing 1 ng/ml leptomycin B. The worms were then recovered and examined for GFP fluorescence.
The protein A beads were then recovered by centrifugation and repeated washes with the same incubation buffer to remove excess anti-Tip110 antibody.
Immunoprecipitates were then recovered by centrifugation and washed three times in 1% Chaps buffer (0.25% for Bmf coimmunoprecipitations). Immunoprecipitates, total cell extracts (40 μg) and subfractionation lysates (30 μg) were separated on NuPage 10% Bis-Tris gels.
The beads were then recovered by centrifugation, and immunoprecipitates were washed three times in cold lysis buffer, and eluted in Laemmli's buffer.
