5: Techniques for studying proteins
- Page ID
- 65999
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- 5.1: Cell Disruption
- There are several ways to break open cells. Whatever method is employed, the crude lysates obtained contain all of the molecules in the cell, and thus, must be further processed to separate the molecules into smaller subsets, or fractions.
- 5.2: Fractionation
- Fractionation of samples typically starts with centrifugation. Using a centrifuge, one can remove cell debris, and fractionate organelles, and cytoplasm. For example, nuclei, being relatively large, can be spun down at fairly low speeds. Once nuclei have been sedimented, the remaining solution, or supernatant, can be centrifuged at higher speeds to obtain the smaller organelles, like mitochondria. Each of these fractions will contain a subset of the molecules in the cell.
- 5.4: Electrophoresis
- Electrophoresis uses an electric field applied across a gel matrix to separate large molecules such as DNA, RNA, and proteins by charge and size. Samples are loaded into the wells of a gel matrix that can separate molecules by size and an electrical field is applied across the gel. This field causes negatively charged molecules to move towards the positive electrode. The gel matrix, itself, acts as a sieve, through which the smallest molecules pass rapidly, while longer molecules are slower-movi