User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/08/25

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Objective

  • Prepare amylose resin column for purification
  • Follow periplasmic protein purification protocol for pMAL-pIII in ER2566

Bench work

  1. Added 10mL of amylose resin to column
    • Ran 3 column volumes of water, then 5 column volumes of column buffer (20mM TrisCl pH 7.4 with 0.2M NaCl and 1mM EDTA)
  2. To the approximately 120mL of pMAL-pIII in ER2566 (resuspended in 30 mM TrisCl pH 8.0 + 20% sucrose) was added 500μL of 0.2M EDTA
    • Centrifuged in four separate 50mL centrifuge tubes; 10 minutes at 10,000xg and 4°C
    • Resuspended each of the four pellets by vigorous pipetting in 25mL of 5mM MgSO4; kept the tube on ice and swirled frequently
    • When evenly resuspended, centrifuged again for 10 minutes at 10,000xg and 4°C
    • Poured all supernatants into one tube → this should be the contents of the periplasm, where the MBP is bound
      • Added 2mL of column buffer to the tube
  3. Using a pump, ran the contents of the periplasm tube through the amylose resin at 1mL/min, collecting 8mL fractions in disposable glass test tubes
    • Followed with 10 column volumes of column buffer and then 10 column volumes of elution buffer (column buffer + 10mM maltose), collecting fractions throughout; still running at 1mL/min
  4. Transformation of ligations from yesterday



Results

Bradford (by eye)

Bradfordbyeye8262011.jpg

  • Cells 1A through 2F are the flow-through from the periplasm/supernatant
  • Cells 2G through 4D are the column wash fractions
  • Cells 4E through 6B are the elution fractions
  • 4F and 4G were the elution fractions with highest apparent protein concentration with likelihood of being MBP