鼠肝细胞溶质的制备

These protocols should yield enough cytosol and organelles for 1-200 MT/Organelle motility assays.
Solutions and Reagents

•  Freshly removed or flash frozen rat liver
•  PBS
• Homogenization Buffer
• Homogenization Buffer containing 0.5 mM mM MgGTP
•  2.3 M sucrose (in Homogenization Buffer containing 0.5 mM MgGTP)
•  Bradford reagent and protein standards
•  PM Buffer containing 0.25 M sucrose.

Equipment

•  20 ml glass homogenizer equipped with Teflon pestle
•  Homogenizer or Drill press
• Superspeed centrifuge (Sorvall RC-5B) with the equivalent of Sorvall SS-34 rotors
• 50 ml polycarbonate centrifuge tubes (Sorvall # 03146)
•  Ultracentrifuge (Beckman L7-55) with the equivalents of Beckman 50.2Ti and SW-28 rotors
• 31.5 ml thick-walled polycarbonate ultracentrifuge tubes (Beckman # 336091) with screw caps (Beckman # 338906)
• 25 ml Ultraclear ultracentrifuge tubes (Beckman # 344058)

Preparation of Rat Liver Cytosol

1. Chill the superspeed centrifuge, ultracentrifuge, SS-34 and 50.2Ti rotor to 4oC.
2. In the cold room, rinse the liver well in PBS, then rinse in freshly prepared Homogenization buffer.
3. Weigh the liver, return to the cold room, and finely mince the tissue with scissors.  Transfer the minced tissue to a glass homogenizer, add 1 volume of homogenization buffer containing  0.5 mM MgGTP.  Keeping the homogenizer immersed in slushy ice, homogenize with a Teflon pestle by 6 slow passes at 3,000 rpm.
4. Transfer the homogenate to 31.5 ml centrifuge tubes, and remove cellular debris by centrifugation at 10,000 x g (9000 rpm) for 10 min at 4oC in a SS-34 rotor.
5. In the cold room, collect the supernatant, transfer it to 31.5 ml ultracentrifuge tubes, pair them by weight, and clarify the cytosol by centrifugation at 100,000 x g (29,000 rpm) for 60 min at 4oC in a 50.2Ti rotor.
6. Collect the clarified cytosolic supernatant, disburse into 50 ul aliquots, and freeze by immersion in liquid nitrogen.  Store at -80oC until use.

Preparation of Rat Liver Organelle Fractions.

1. Perform steps 1-4 as in "Preparation of Rat Liver Cytosol" above except in step 2, homogenize the minced liver in homogenization buffer containing  0.5 mM MgGTP and 0.25 M sucrose.
2. In the cold room, collect the supernatant (clarified homogenate) and add 2.3 M sucrose stock to make the final concentration 1.25 M sucrose.
3. Set up a sucrose density step gradient in a 25 ml ultraclear ultracentrifuge tube.  All sucrose solutions should be made by dilution of the 2.3 M sucrose stock solution with homogenization buffer containing 0.5 mM MgGTP and chilled to 4oC.  Add a 2 ml 2.0 M sucrose cushion to the bottom of the tube. Being careful not to disturb the cushion, layer 12 ml of clarified homogenate containing 1.25 M sucrose on the 2.0 M sucrose by using a pipette and allowing the solution to slowly run in a steady stream down the side of the centrifuge tube.  Then add a 12 ml layer of 1.1 M sucrose, and finally an 8 ml layer of 0.25 M sucrose.  Discard any excess homogenate.  Pair the gradient by weight with a balance tube.
4. Separate the organelles by density by centrifuging the gradient at 100,000 x g (28,000 rpm) for 3 h at 4oC in a SW-28 rotor.
5. In the cold room, with a Pasteur pipette carefully harvest the off-white band of Golgi membrane at the 0.25 M/1.1M sucrose interface and the off-white band of ER (ER) membrane at the 1.1 M/1.25 M sucrose interface and place them in separate 31.5 ml ultracentrifuge tubes on ice.
6. Perform a Bradford assay to determine protein concentration of the Golgi and ER membrane fractions.
7. Dilute both membrane fractions with 3 volumes of 0.25 M sucrose in homogenization buffer containing 0.5 mM GTP, and pellet the membranes by centrifugation at 100,000 x g (29,000 rpm) for 1 h at 4oC in a 50.2Ti rotor.
8. In the cold room, discard the supernatants and separately resuspend the dense, sticky ER and Golgi membrane pellets by extensive trituration in PM buffer containing 0.25 M sucrose and 0.5 mM GTP  to achieve a protein concentration of 5 mg/ml.
9. Disburse into 20 ul aliquots and freeze by immersion in liquid nitrogen.  Store at -80oC until use.
10. Determine the proper dilution of ER of Golgi membranes for use in the MT/Organelle motility assay.
    View various dilutions (in PM buffer) of membranes in simple perfusion chambers by VE-DIC microscopy.  Note the dilution required so that ~20% of the area of the microscopic field is covered with organelles.  For setting up the membrane MT motility assay, you will make need a stock that is 6 x the dilution just determined, which will be called 6X MEMBRANES.