Purify the protein that Tamra expressed last week Link to her notebook page from that day.
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.
- Thaw resuspended cells
- 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.
- Centrifuge lysed cells: 2 hours at 18000 rpm and 4C.
- Filter supernatant in filter flask. (syringe filters don't quite work well enough)
- Perform buffer exchange on FPLC with 26/10 desalting column.
- Buffer: 25mM Tris, 50mM NaCl pH 8
- We had about 150mL of protein solution. Buffer exchange took a long time.
- Stress importance of small buffer amounts when resuspending.
- Store protein in fridge overnight.
- Add data and results here...
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