Purpose
- Extract protein from cells as part of protein purification.
Procedure
- Cells were taken out of -80C freezer and placed in ~30C warm water for cell thawing.
- Once thawed, cells were sonicated and incubated on ice for each 30 second intervals. This process is repeated 3 times.
- Once sonicated, cells were placed in centrifuge tubes and in centrifuge for 2 hours under 18,000 rpm and 4C.
- Once centrifuged, supernatant were kept, filtered with 450mm filter paper, and kept in 4C until the next day.
Discussion
- Cells taken from the freezer were complete cells. After sonication, cells were burst open. The organelles, plasma membrane, and internal proteins were all exposed in solution. Centrifugation allowed the heavier organelles and plasma membranes to sink to the bottom of the tube, forming a pellet, and remaining proteins or other lighter biological molecules in supernatant. This is an effective and simple way to purify proteins from prokaryotic cells.
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Experimental Biological Chemistry I
