Blood Cell Fractionation Lab Blood is composed of 2 main fractions: the plasma, or liquid part of the blood, and the blood cells themselves. These two components contain very different proteins and this lab will demonstrate several different mechanisms used to isolate various extracellular and cellular fractions. The most common and important protein in red bood cells (by far the most common cell in blood) is hemoglobin which is composed of a protein portion (globin) and a prosthetic group (heme bound to iron). We will use the metal binding affinity of iminodiacetic acid (IDA) attached to agarose beads to specifically remove hemoglobin from the cytoplasmic fraction of red blood cells and elute it from the IDA beads using the strong metal binding salt ethylenediaminetetraacetic acid (EDTA). Next week we will separate out the proteins from the various fractions using SDS-PAGE and study the oxygen binding capacity of hemoglobin spectroscopically. 1) Spin down 0.5 mL of fresh horse blood at 3000x g (5000 rpm) in a benchtop centrifuge for 2 minutes. We will not use human blood to limit the danger of blood borne pathogens. 2) Remove the slightly yellowish supernatant above the compacted red blood cells and label it as plasma. Store this tube at -20 oC until next week. 3a) Gently resuspend the red blood cells in 1.0 mL of PBS (phosphate buffered saline). 3b) Spin down the red blood cells again at 3000x g in a benchtop centrifuge for 2 minutes and discard the supernatant. 3c) Repeat 3a and 3b twice for a total of 3 washes. This will remove any plasma proteins that are sticking the blood cells. 4) Resuspend the blood cells in 0.5 mL deionized water and heat to 55 oC for 5 minutes in a heating block. The change in ionic strength and heating the sample lyses (breaks open) the blood cells and releases the cytoplasmic proteins into the solution. 5) Spin down the membranes at 12,000x g (12,000 rpm) for 5 minutes. 6) Remove the red supernatant from the tube and place it in a fresh tube, but keep the membrane pellet also! The membrane pellet will be used later. Put 100 of the red supernatant into a different tube, label it cytoplasm, and put it with your plasma fraction in the -20 oC freezer. 7) Put 100 of your red supernatant into a fresh tube and add 100 of IDA agarose beads (50% slurry, so approx. 50 of beads). Put this mixture on the shaker or rotator for 15 minutes. Put the remainder of your red supernatant in the -20 oC freezer until next week when we study the chemical difference in hemoglobin when bound to O2 or CO2! 8) While the supernatant is shaking, resuspend your membrane pellet in 1.0 mL of distilled water and vortex for at least 15 seconds. Spin down the membrane at 14,000x g for 10 minutes and discard this supernatant. Warning: the membrane pellet at this step is hard to distinguish! 9) Repeat step 8 twice for a total of 3 washes. The membrane pellet should now be pinkish with a clear supernatant. Place this membrane fraction in the -20 oC freezer with your other tubes. 10) Retrieve your cytoplasm + beads tube from step 7 and centrifuge it at 5000 rpm for 1 minute. The red color (hemoglobin) should be primarily sticking to the IDA beads. 11) Remove the supernatant and add 0.5 mL of PBS to the beads. Vortex briefly to mix the beads, then centrifuge again at 5000 rpm for 1 minute. 12) Repeat step 11. This washes unbound proteins away from the IDA beads + hemoglobin. 13) Add 100 of 50 mM EDTA to the beads. Vortex briefly, and shake or rotate the mixture for 10 minutes. 14) Centrifuge the mixture at 1000 rpm for 1 minute. Remove the red supernatant and place it in a fresh tube labeled hemoglobin. Place the hemoglobin tube in the -20 oC freezer with your other tubes until next week. 15) Resuspend your used IDA beads in 150 of EDTA and take them to the instructor's large tube of beads to be reused.