The purpose of this experiment is to produce a relatively pure fraction of red blood cell plasma membrane from a sample of sheep red blood using differential centrifugation. Once produced, the fraction will be frozen and stored until later in the semester, when it will be analyzed for its protein content using SDS-polyacrylamide gel electrophoresis.
1. Using a 25 ml plastic pipette, transfer 20 ml of whole blood to a 40 ml round bottom centrifuge tube. Balance the tubes with that of another group by simply adding blood to the lighter tube. Place the balanced tubes directly across from one another in the centrifuge. Once the rotor is full, centrifuge the blood at 1,000 x g for 5 minutes at 0-4 C to collect the red blood cells.
2. Using a pasteur pipet, carefully decant the supernatant (plasma) from the tube and discard. Add 20 ml ISOTONIC sodium phosphate buffer and resuspend the red cells by GENTLY inverting the tube five times. This will help wash the red cells of any residual plasma. (Make sure the entire pellet is suspended. You may need to knock the bottom of the tube against the heal of your hand to loosen the entire pellet.). Balance the tubes with that of another group by adding some isotonic buffer to the lighter tube, place the balanced tubes directly across from one another in the centrifuge. Once the rotor is full, centrifuge the blood at 1,000 x g for 5 minutes at 0-4 C to collect the erythrocytes.
3. Decant and discard the supernatant. Add 25 ml HYPOTONIC sodium phosphate buffer. Cap the tubes and vigorously agitate the tubes for 5 seconds every 30 seconds for a period of 5 minutes. This action will help to lyse the cells.
4. Balance the tubes with that of another group by adding hypotonic buffer to the lighter tube, then place the balanced tubes directly across from one another in the centrifuge. This is a high speed centrifugation, so the balance is particularly important in this step. Once the rotor is full, centrifuge the blood at 40,000 x g for 5 minutes at 0-4 C to collect the red cell plasma membranes.
5. With a 10 ml serological pipet, decant the top 25 ml of the deep red supernatants. Be careful not to overfill the pipet and contaminate the pipetting device. Add 25 ml HYPOTONIC buffer to the tube, seal the tubes and invert three times to wash the plasma membrane of residual hemoglobin. Balance the tubes with that of another group by adding hypotonic buffer to the lighter tube, then place the balanced tubes directly across from one another in the centrifuge. This is another high speed centrifugation, so the balance is particularly important in this step. Once the rotor is full, centrifuge the blood at 40,000 x g for 5 minutes at 0-4 C to collect the red cell plasma membranes.
6. At this point you should be able to distinguish the pellet from the supernatant. Repeat step 5 if time permits. You can decant the supernatant with a pasteur pipet at this point.
7. Following the final decant, add 3 ml ISOTONIC buffer to each pellet, swirl to resuspend the pellets, transfer the suspension of plasma membranes to a freezing tube and freeze the suspension for future use.