Isoelectric focusing properties

There are three distinct modes of electrophoresis zone electrophoresis, isoelectric focusing, and isotachophoresis. These three methods may be used alone or in combination to separate molecules on both an analytical (p.L of a mixture separated) and preparative (mL of a mixture separated) scale. Separations in these three modes are based on different physical properties of the molecules in the mixture, making at least three different analyses possible on the same mixture. 

Two-dimensional electrophoresis is normally run so that proteins are separated from each other on the basis of a different molecular property in each dimension. The most commonly utilized method entails separation of proteins by isoelectric focusing (see below) in the first dimension, with separation in the second dimension being undertaken in the presence of SDS, thus promoting band separation on the basis of protein size. Modified electrophoresis equipment that renders two-dimensional electrophoretic separation routine is freely available. Application of biopharmaceuti-cal finished products to such systems allows rigorous analysis of purity. 

As with SDS-PAGE gel methods, gel-based isoelectric focusing (lEF) methods have been used for decades to determine isoelectric point pi), which is an intrinsic property of protein molecules. Some complex proteins have multiple charge isoforms with multiple isoelectric points. These isoforms are separated as multiple bands in the lEF gel method. However, like other gel method, the lEF gel has limitations it is not automated, not reproducible, and not quantitative for pi determination. It is also labor intensive and requires large volumes of toxic reagents for staining. 

D electrophoresis is normally run so that proteins are separated from each other on the basis of a different molecular property in each dimension. The most commonly utilized method entails separation of proteins by isoelectric focusing (see below) in the first dimension, with... 

Understand how proteins act as polyelectrolytes discuss separation methodologies based on ionic properties of proteins electrophoresis, ion-exchange chromatography, and isoelectric focusing. 

The net charge on a protein is the algebraic sum of all its positive and negative charges. There is a specific pH for every protein at which the net charge it carries is zero. This isoelectric pH value, termed the isoelectric point, or pi, is a characteristic physicochemical property of every protein. The definition of pi for molecules as complex as proteins is more or less an operational one and is taken to be that pH at which a protein has zero electrophoretic mobility in an isoelectric focusing run. Nevertheless, it has been shown that the pis of some acidic proteins (up to about pH 7) can be calculated from their amino acid compositions.3 5... 

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