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

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Objective

  1. 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 450nm holes.
  6. After the two hours of centrifuging were completed, the cells were filtered using vacuum filtration and membrane-filter filter paper with 450nm holes. This separated the protein from the rest of the cell organelles. \
  7. The proteins were then collected in Falcon tubes and refrigerated over night.