The purpose of this experiment is to produce a relatively pure fraction of mammalian hepatocyte nuclei from a sample of freshly homogenized rat liver using differential centrifugation. Once produced, the fraction will be frozen and stored until later in the semester.
A. Tissue Preparation. 1. On the ice-cold stage, mince the liver into 0.5 cm3 pieces with single-edge razor blases. Once completed, pool your liver with the group across from you and transfer it to a glass homogenization mortar. Then add enough homogenization buffer (lysis buffer) to reach the bottom of the neck of the mortar.
B. Homogenization 1. Homogenize the minced tissue under the following conditions: a, type of homogenizer: Potter-Elvehjem homogeniser, b, speed and duration: maximum speed, 8 strokes of the pestle, c, Homogenization medium: 0.25 M Standard Sucrose Buffer (0.25 M SSB). 2. Pour the homogenate into an ice-cold erlenmeyer flask through 4 layers of cheesecloth to filter any clumps of connective tissue or unhomogenized chunks of tissue. Pool your filtered homogenate with the other group at your table.
C. Prepare the Centrifuge Tube 1.Using a 10 ml serological pipette, carefully add 24 ml of 2.0 M Standard Sucrose Buffer (2.0 M SSB) to the very bottom of a 30 ml centrifuge tube. 2. Using a 5 ml serological pipette, carefully layer 5 ml of the homogenate onto the 2.0 M SSB.3. Carefully place your centrifuge tube on ice and carry it to the centrifuge room.
E. Centrifugation: 1. Carefully slide your centrifuge tube into a centrifuge tube holder. Do not disrupt the sucrose gradient. 2. Balance your tube (w/ holder) with another tube. To balance the two, simply remove homogenate from the heavier tube using a pasteur pipette. Please be careful here, you do not want to disrupt the sucrose gradient. 3. Place the tubes directly across from one another on the SW 28 rotor, place the rotor in the ultracentrifuge, and spin at 23,000 RPM for 30 minutes in an ultracentrifuge.
Following centrifugation, decant and dispose of the supernatant. Add 2 ml of freezing medium to the tube, resuspend the pellet, then transfer the pellet to a freezing vial. Freeze the sample at -60 C.