3: Methods of Protein Purification and Characterization
- Page ID
- 165270
\( \newcommand{\vecs}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} } \) \( \newcommand{\vecd}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash {#1}}} \)\(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\) \(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\)\(\newcommand{\AA}{\unicode[.8,0]{x212B}}\)
- 3.1: Protein Purification
- A successful protein purification procedure can be nothing short of amazing. Whether you are starting off with a recombinant protein which is produced in E. coli, or trying to isolate a protein from some mammalian tissue, you are typically starting with gram quantities of a complex mixture of protein, nucleic acids, polysaccharide, etc. from which you may have to extract milligram (or microgram!) quantities of desired protein at high purity, and hopefully with high yield.
- 3.2: Cell Disruption
- Cell disruption is a method or process for releasing biological molecules from inside a cell. 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.
- 3.3:Cell Fractionation and Centrifugation
- 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.
- 3.4: Chromatography
- Chromatography is the most discriminating analytical technique used to separate mixture of a proteins on the basis of size, affinity or ionic interaction.
- 3.5: Electrophoresis
- Electrophoresis is used in laboratories to separate macromolecules based on size. The technique applies a negative charge so proteins move towards a positive charge. Electrophoresis is used extensively in DNA, RNA and protein analysis.