Ampholytes isoelectric point

A large part of the dissolved amino acid exists as the ampholyte (2witterion). The isoelectric point (pi) is the pH at which the net electric charge of a dissolved amino acid molecule is 2ero. p /is expressed as... 

Because protein samples are actually ampholytes, when samples are loaded onto the gel and a current is appHed, the compounds migrate through the gel until they come to their isoelectric point where they reach a steady state. This technique measures an intrinsic physicochemical parameter of the protein, the pi, and therefore does not depend on the mode of sample appHcation. The highest sample load of any electrophoretic technique may be used, however, sample load affects the final position of a component band if the load is extremely high, ie, high enough to titrate the gradient ampholytes or distort the local electric field. 

Isoelectric focusing takes along (from ca 3 to 30 h) time to complete because sample compounds move more and more slowly as they approach the pH in the gel that corresponds to their isoelectric points. Because the gradient ampholytes and the samples stop where they have no mobiHty, the resistivity of the system increases dramatically toward the end of the experiment, and the current decreases dramatically. For this reason, isoelectric focusing is usually mn with constant voltage. Constant current appHcation can lead to overheating of the system. 

An important quantity characterizing the amino acids, peptides and proteins is the isoelectric point, which is the value of pH at which the ampholytes do not migrate in an electrical field. This occurs when... 

Following similar reasoning, the adsorption pattern observed for ampholytic polyelectrolytes Ocan be explained. As illustrated in Fig. 4, polyampholytes show maximum adsorption around their isoelectric point (i.e., the pH where the net charge of the polyampholyte is zero). 

An advantage of CIEF is that the total length of the capillary is filled with sample and ampholyte mixture thus, larger quantities of proteins can be separated. It therefore can be advantageously used as a sample preconcentration step prior to capillary gel electrophoresis. Furthermore, CIEF is a suitable and very precise method for measuring the isoelectric point of biomolecules. 

Isoelectric focusing is a procedure used to determine the isoelectric point (pi) of a protein (Fig. 3-21). A pH gradient is established by allowing a mixture of low molecular weight organic acids and bases (ampholytes p. 81) to distribute themselves in an electric field generated across the gel. When a protein mix-... 

FIGURE 3-21 Isoelectric focusing. This technique separates proteins according to their isoelectric points. A stable pH gradient is established in the gel by the addition of appropriate ampholytes. A protein mixture is placed in a well on the gel. With an applied electric field, proteins enter the gel and migrate until each reaches a pH equivalent to its pi. Remember that when pH = pi, the net charge of a protein is zero. 

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