Cell Biology, Fall 2008
Lab Exercise: Protein Extraction and Purification
Proteins are relatively large organic compounds made of amino acids arranged in a linear chain and joined together between the carboxyl atom of one amino acid and the amine nitrogen of another. This bond is called a peptide bond. The sequence of amino acids in a protein is defined by a gene and encoded in the genetic code. There are twenty different amino acids, each with their own properties.
Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of all living organisms and participate in every process within cells. Many proteins are enzymes that catalyze ...view middle of the document...
Objectives of exercise:
â€¢ Isolation of proteins by â€œsalting outâ€
â€¢ Filtration by negative pressure
â€¢ Separation of salt from protein by dialysis
General. Almost all the molecules a cell makes or has are composed of carbon atoms bonded to one another. Carbon is unequalled in its ability to form large, diverse molecules. Next to water, compounds containing carbon are the most common substances in living organisms. The most common of these macromolecules are carbohydrates (sugars, starch, and glycogen, and cellulose) proteins (which serve structural, catalytic and signaling functions), lipids, and nucleic acids. The proportion of these macromolecules in a cell is dependent on the type and function of the particular cell. To study any one of these macromolecules in a cell, the components must be selectively isolated and separated.
There are a variety of ways to separate components of a mixture. Methods of separation take advantage of differences in a moleculeâ€™s physical and chemical properties. Some common methods for separation in the lab are centrifugation, chromatography, dialysis, electrophoresis, and salting out. These methods can be used in combination to isolate and purify, to some degree, specific types of macromolecules.
Methods for separation and purification. Dialysis is the use of a semi-permeable membrane to sort molecules according to size. The size of the pores of the membrane determines what is held back and what goes through. This is the basis of dialysis machines used in hospitals and our understanding of how kidneys work. Centrifugation is the use of centrifugal force to separate molecules according to mass. Dense particles will spin out to the bottom of a centrifuge; less dense particles will not fall to the bottom and in some cases will float to the top as is the case for fats or lipids. Salting out is the use of solutions containing dissolved salts to form a molecular aggregation with protein. The salt-protein complex will precipitate out of solution. The concentration of salt required for precipitation of a specific protein depends on the particular protein and the pH of the solution â€“ i.e. the charge of the protein complex. The salts often used are from heavy metals (e.g., HgCl2, AgNO3, and CuO4); certain acids; and concentrated solution of ammonium sulfate, sodium sulfate, and sodium chloride. Solvents used are usually ethyl and methyl alcohol. Chromatography separates pigments or other molecules based on mass and solubility. Electrophoresis separates molecules according to charge and size
Protein purification. In order to perform analyses, a protein must be purified away from other cellular components. This process usually begins with cell lysis, in which a cell's membrane is disrupted and its internal contents released into a solution known as a crude lysate. The resulting mixture can be purified using ultracentrifugation, which fractionates...