User:Matt Hartings/Notebook/Photosynthesis/2012/06/18

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Objective

Purify the protein that Tamra expressed last week Link to her notebook page from that day.

Description

Tamra had made up 3 tubes containing the resuspended cells and had stored them over the weekend at -20. I wanted to do extraction and purification on same day or as soon as possible to cut down on any protease activity.

  1. Thaw resuspended cells
  2. Lyse the cells with the sonicator. Power level 12. 30 seconds of sonication followed by 30 seconds on ice. Repeat for a total of three cycles.
  3. Centrifuge lysed cells: 2 hours at 18000 rpm and 4C.
  4. Filter supernatant in filter flask. (syringe filters don't quite work well enough)
  5. Perform buffer exchange on FPLC with 26/10 desalting column.
    1. Buffer: 25mM Tris, 50mM NaCl pH 8
    2. We had about 150mL of protein solution. Buffer exchange took a long time.
    3. Stress importance of small buffer amounts when resuspending.
  6. Store protein in fridge overnight.

Data

  • Add data and results here...

Notes

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