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
- 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.
- 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
- Chill the superspeed centrifuge, ultracentrifuge, SS-34 and 50.2Ti rotor to 4oC.
- In the cold room, rinse the liver well in PBS, then rinse in freshly prepared Homogenization buffer.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Perform a Bradford assay to determine protein concentration of the Golgi and ER membrane fractions.
- 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.
- 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.
- Disburse into 20 ul aliquots and freeze by immersion in liquid nitrogen. Store at -80oC until use.
- 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.
*=As published in...
Waterman-Storer, C.M. (In press) Microtubule/organelle motility assays. In: Current Protocols in Cell Biology, J.S. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott-Schwartz, and K.M. Yamada, eds. John Wiley, NY.