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45,000

15,000

Note: All data represent the status of the sample after the designated procedure has been carried out. Activity and specific activity are defined on page 94.

Note: All data represent the status of the sample after the designated procedure has been carried out. Activity and specific activity are defined on page 94.

somewhat empirical, and many protocols may be tried before the most effective one is found. Trial and error can often be minimized by basing the procedure on purification techniques developed for similar proteins. Published purification protocols are available for many thousands of proteins. Common sense dictates that inexpensive procedures such as salting out be used first, when the total volume and the number of contaminants are greatest. Chromatographic methods are often impractical at early stages, because the amount of chro-matographic medium needed increases with sample size. As each purification step is completed, the sample size generally becomes smaller (Table 3-5), making it feasible to use more sophisticated (and expensive) chromatographic procedures at later stages.

Proteins Can Be Separated and Characterized by Electrophoresis

Another important technique for the separation of proteins is based on the migration of charged proteins in an electric field, a process called electrophoresis. These procedures are not generally used to purify proteins in large amounts, because simpler alternatives are usually available and electrophoretic methods often adversely affect the structure and thus the function of proteins. Electrophoresis is, however, especially useful as an analytical method. Its advantage is that proteins can be visualized as well as separated, permitting a researcher to estimate quickly the number of different proteins in a mixture or the degree of purity of a particular protein preparation. Also, electrophoresis allows determination of crucial properties of a protein such as its isoelectric point and approximate molecular weight.

Electrophoresis of proteins is generally carried out in gels made up of the cross-linked polymer polyacryl-amide (Fig. 3-19). The polyacrylamide gel acts as a molecular sieve, slowing the migration of proteins approximately in proportion to their charge-to-mass ratio. Migration may also be affected by protein shape. In elec-trophoresis, the force moving the macromolecule is the electrical potential, E. The electrophoretic mobility of the molecule, is the ratio of the velocity of the par ticle molecule, V, to the electrical potential. Electrophoretic mobility is also equal to the net charge of the molecule, Z, divided by the frictional coefficient, f, which reflects in part a protein's shape. Thus:

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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