User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/10/02

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Objective

  • To prepare cells for protein purification

Description

  1. The cells were removed from the -80°C freezer and placed in an warm water bath until thawed.
  2. 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.
  3. 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.
  4. The cells were centrifuged for 2 hours at 4°C at 18,000 rpm.
  5. During this process, the binding buffer and elusion buffer were filtered using vacuum filtration and membrane-filter filter paper with 0.45μm holes.
  6. 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.
    1. The pallet that stuck to the centrifuge tube was bleached and transferred into a Falcon tube and thrown in the "Bio Waste" container.
  7. The filtrate was collected in Falcon tubes and stored over night in the refrigerator.

Notes

  • 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.


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