5: Protein Purification
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- 5.2: 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.
- 5.3: 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
- 5.5: Electrophoresis
- DNA molecules are long and loaded with negative charges, thanks to their phosphate backbones. Electrophoretic methods separate large molecules, such as DNA, RNA, and proteins based on their charge and size. For DNA and RNA, the charge of the nucleic acid is proportional to its size (length). For proteins, which do not have a uniform charge, a clever trick is employed to make them mimic nucleic acids.
- 5.6: Gel Exclusion Chromatography
- Gel exclusion chromatography is a low resolution isolation method. This involves the use of beads that have tiny “tunnels" in them that each have a precise size. The size is referred to as an “exclusion limit," which means that molecules above a certain molecular weight will not fit into the tunnels. Molecules with sizes larger than the exclusion limit do not enter the tunnels and pass through the column relatively quickly by making their way between the beads.
- 5.7: Ion Exchange Chromatography
- In ion exchange chromatography, the support consists of tiny beads to which are attached chemicals possessing a charge. Each charged molecule has a counter-ion.
- 5.8: Affinity Chromatography
- Affinity chromatography exploits the binding affinities of target molecules (typically proteins) for substances covalently linked to beads. For example, if one wanted to separate all of the proteins in a sample that bound to ATP from proteins that do not bind ATP, one could covalently link ATP to support beads and then pass the sample through column. All proteins that bind ATP will “stick" to the column, whereas those that do not bind ATP will pass quickly through it.
- 5.10: Histidine Tagging
- Histidine tagging is a powerful tool for isolating a recombinant protein from a cell lysate. The protein produced when this gene is expressed has a run of histidine residues fused at either the carboxyl or amino terminus to the amino acids in the remainder of the protein. The histidine side chains of this “tag" have an affinity for nickel or cobalt ions, making separation of histidine tagged proteins from a cell lysate is relatively easy.
