Tls 55 rotor

6.5 × 10⁷ human PBMC were treated with 40 μM of hTip-CSKH in serum-free RMPI-1640 during 1 hour at 37°C. The cells were washed in medium and the membranes were extracted during 20 min in 200 μL of ice-cold lysis buffer containing Tris 25 mM pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Triton-X100, 1 ug/mL leupeptin, 1 ug/mL pepstatin, 4 ug/mL aprotinin, 1 mM PMSF, 1 mM Na3VO4, and 50 mM NaF. For sucrose density gradients, cell extracts were mixed with 250 μL of 80% sucrose in TNE buffer (Tris 25 mM pH 7.4, 150 mM NaCl, and 2 mM EDTA) and overlayed with 730 μL of 35% sucrose and 320 μL 5% sucrose and centrifuged at 200,000 g during 5 h, 4°C (Beckman Optima, rotor TLS-55). Eight fractions of equal volume were recovered from the top of the gradient, diluted with 1 mL of TNE, and detergent resistant membranes in each fraction were precipitated by ultracentrifugation at 100,000 g during 45 min, 4°C. The pellets were extracted in 20 μL of Laemmli buffer: half used for WB and Flotillin-2 detection and the other half for SDS-PAGE in Tris-Tricine buffer for peptide identification as described [42 (link)]. To facilitate the identification of the peptides in this system, erythrocyte membranes were prepared in parallel and incubated with the different peptides used. Peptides were identified by sample separation in Tris-Tricine SDS-PAGE.

Thirty percent Percoll was prepared from a 100% Percoll solution by mixing 100% Percoll with 1xPBS at a ratio of 3:7 (volume). A total of 2 mL of the 30% Percoll solution was pipetted into a centrifugation tube. Total microsomes (P100k) resuspended in PBS or samples eluted from the immunoprecipitated beads (200 μL) were carefully layered on top of the Percoll solution. Centrifugation was performed at 95,000 x g (rotor TLS55, Beckman) at 4°C for 40 min. After centrifugation, the gradient was fractionated into 21 tubes, 100 μL per fraction. The samples were then analyzed by Western blotting and quantified with ImageJ.