Cell Lysis Protocol for Protein Isolation
Small proteins can be prepared synthetically, but for many reasons there is no synthetic machinery better than the cell itself. Typically, the first step in protein isolation is cell lysis.
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Cell Lysis
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In cell lysis, the membrane around the cell ruptures. The word "lysis" comes from a Greek root meaning "to loosen." In cell lysis, the forces holding the cell membrane together are disrupted, causing it to rupture. The contents of the cell are then released into the surrounding solution, including the protein of interest. Usually this is done by the addition of a chemical solution called lysis buffer.
Preparing Lysis Buffer
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The choice of lysis buffer will depend on the cell type, the protein and the application. A myriad of different lysis solutions are available commercially, or common recipes can be used to prepare lysis buffer from common chemicals. The best buffer to use will vary depending on the cell type used, the protein to be isolated and the final application, but, in general, a lysis solution contains a detergent molecule (often NP-40 or Triton-X) that dissolves the cell membrane, along with buffers, chelators and protease inhibitors to prevent the protein from being damaged once released from the protective environment of the cell.
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Lysis Conditions
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Performing lysis under ice-cold conditions prevents damage to the protein of interest. Lysis buffer is usually used ice-cold, and the lysing process is allowed to proceed under cold conditions. The solution is gently rocked to encourage even dispersion of the cells and lysis products. After about 5 to 10 minutes, the lysis solution is usually neutralized to prevent further activity. These precautions reduce damage to the protein and reduce amount of extraneous debris produced that will later have to be removed.
Other Lysis Methods
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Mechanical force can also be used to lyse cells. Although chemical lysis is the gentlest and most commonly used, harsher mechanical methods for lysing cells can be used in some cases. These include sonication, repeated freeze-thaw cycles and homogenation, which is a mashing process. Consult the technical literature to find the most appropriate method for your specific case.
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