- To prepare cells for protein purification
- The cells were removed from the -80°C freezer and placed in an warm water bath until thawed.
- After the cells were thawed, they were sonicated (using a Misonix XL-2000 series Ultrasonic Liquid Processor)for 30 seconds and then placed in an ice bucket for 30 seconds. This process was repeated for a total of three times to blast open the cells.
- The opened cells were then poured into a 35mL centrifuge tubes. Water was filled in a second centrifuge tube such that the mass of the second tube was within 0.01 grams of the first tube, making them balanced.
- The cells were centrifuged for 2 hours at 4°C at 18,000 rpm.
- During this process, the binding buffer and elusion buffer were filtered using vacuum filtration and membrane-filter filter paper with 0.45μm holes.
- After the two hours of centrifuging were completed, the cells were filtered using vacuum filtration and membrane-filter filter paper with 0.45μm holes. This separated any large clumps from the proteins and rest of the solution.
- The filtrate was collected in Falcon tubes and stored over night in the refrigerator.
- The cells need to be removed immediately after centrifugation because the large, heavy clumps that would likely stick to a protein purification column are in the pallet. The longer the centrifuged tube sits after being spinned down, the more likely the larger particles will go back into the protein-buffer solution, which makes it difficult to filter.