Complete mini edta free inhibitor

Vesicle IP and flotation assays were performed as previously described (Zhao et al., 2001 (link); Tanaka et al., 2016 (link)). WT and Kif1bβ^GFP/GFP adult mouse brains were homogenized with ∼5 ml Hepes-sucrose buffer (10 mM Hepes, pH 7.4, 320 mM sucrose, 5 mM MgSO₄, 1 mM EGTA, and protease inhibitors [cOmplete mini EDTA-free inhibitor; Roche]) and cleared by centrifugation twice at 1,000 g for 10 min at 4°C. For IP, the supernatant (S1) was mixed with 50 µl magnetic beads (μMACS Protein A; 130-042-601; Miltenyi Biotec) and 2 µg anti-GFP antibody for 2 h at 4°C. The beads were washed, eluted, and sampled for IB. For the flotation assay, the supernatant was diluted in 60% Nycodenz at a volume ratio of 1:5 and subjected to step-gradient ultracentrifugation with Nycodenz (0, 10, 20, 30, 40, 50, and 60%; Progen Biotechnik) in OptiSeal tubes (11.2 ml; 362181; Beckman Coulter) using an Optima XL-100K Ultracentrifuge with an NVT 65 rotor (Beckman Coulter) at 65,000 rpm for 2.5 h at 4°C with both acceleration and deceleration in the slowest mode of 9. The effluent fractions were collected by piercing the bottom of the tube with a 22G needle (NN-2238R; Terumo) and placing in 1.5-ml tubes at 500 µl/tube, and the fractions were subjected to IB.