A Liver

1. A Liver B Brain Livers of 3 to 5 mice Brains of 3 to 5 mice • 600×gmax for 10 min, 4 °C • 1,000×gmax for 10 min, 4 °C • Pellet is resuspended twice in buffer A at 600×gmax for 10 min, 4 °C pPNS N • Resuspension of pPNS in buffer A at 3,500×gmax for 10 min, 4 °C • Resuspension of HMP in buffer A at 3,500×gmax for 10 min, 4 °C N pPNS • Resuspension of pPNS in buffer A at 3,500×gmax for 10 min, 4 °C • Resuspension of HMP in buffer A at 5,000×gmax and 5,500×gmax respectively, for 10 min, 4 °C HMP pPMS • 25,000×gmax for 20 min, 4 °C HMP pPMS • 25,000×gmax for 30 min, 4 °C LMP 25,000×gmax Supernatant 25,000×gmax Supernatant 1 • Resuspension in 1.5ml of buffer D LMP 1 IF + UL („Myelin“) Sucrose gradient sLMP • Resuspension in buffer B to a final volume of 4.5 ml and placing at the bottom of a 10 ml polycarbonate tube • Overlay with 4.5 ml of buffer A • 107,000×gmax for 45 min, 4 °C • Gradient loading (see right) • 70,000×gmax for 120 min, 4 °C with slow acceleration and deceleration • sample harvesting (see materials and methods) Pellet + LL Gradient loading: • 1.5 ml of sLMP • 6 ml of 20 % to 40 % Nycodenz™ continuous density gradient • 1 ml of 60% Nycodenz™ density cushion • Resuspension in buffer C • 25,000×gmax for 30 min, 4 °C Final gradient harvested in eight fractions & Interphase LMP 2 25,000×gmaxSupernatant 2 • Resuspension in 1.5 ml of buffer D sLMP 2 Gradient loading: • 1.5 ml of sLMP 2 • 6 ml of 15 % to 30 % Nycodenz™ continuous density gradient • 1 ml of 60% Nycodenz™ density cushion • Gradient loading (see right) • 116,000×gmax for 75 min, 4 °C with slow acceleration and deceleration • sample harvesting (see materials and methods) Final gradient harvested in eight fractions