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Protein Separation by Isoelectric point

The first-dimension separation of 2-D and stops migrating (Figure 3.2). If, by diffusion, it electrophoresis is IEF, where proteins are drifts away from the point in the gradient separated on the basis of differences in their pI. corresponding to its pI, it acquires charge and is The pI of a protein is the pH at which it carries no pulled back. In this way, proteins condense, or net charge, and it is a characteristic that is are focused, into sharp bands in the pH gradient determined by the number and types of charged at their characteristic pI values. groups the protein carries. IEF proceeds until a steady state is reached. Proteins are amphoteric molecules, which carry a Proteins approach their pI values at different rates positive, negative, or zero net charge depending but remain relatively fixed at those pH values for on the pH of their environment. For every protein, extended periods. This is in contrast to there is a specific pH at which its net charge is conventional electrophoresis (for example, zero (its pI). Proteins show considerable variation polyacrylamide gel electrophoresis, or PAGE), in pI, though pI values usually fall in the range of where proteins continue to move through the pH 3–12, with the majority falling between pH 4 medium until the electric field is removed. and pH 8. A protein is positively charged at pH Moreover, in IEF, proteins migrate to their steady- values below its pI and negatively charged at pH state positions from anywhere in the system. values above its pI (Figure 3.1). IEF for 2-D electrophoresis is performed under For IEF, a protein is placed in a medium with a pH denaturing conditions so that proteins are gradient and subjected to an electric field. In completely disaggregated and all charged groups response to the field, the protein moves toward are exposed to the bulk solution. Consequently, the electrode with the opposite charge. Along the resolution is best under denaturing conditions. way, it either picks up or loses protons. Its net Complete denaturation and solubilization are charge and mobility decrease until the protein required to minimize aggregation and eventually arrives at the point in the pH gradient intermolecular interactions, thus ensuring that equal to its pI. There, the protein is uncharged each protein is present in only one configuration.

Fig. 3.1. Dependence of protein net charge on the pH of its environment. The pH at which the net charge is 0 is the i Net Charge soelectric point (pI).

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Fig. 3.2. Principle of IEF. A mixture of proteins is separated in a pH gradient and within an electric field according to each protein’s pI and independently of its size. The proteins migrate until they reach their pI.

IEF Media: IPG Strips vs. Carrier Ampholytes IEF for 2-D electrophoresis is most commonly Historically, first-dimension IEF was performed performed using immobilized pH gradient (IPG) using carrier ampholyte–generated pH gradients strips. As their name implies, IPG strips contain and tube gels. This type of first dimension has buffering groups covalently bound to a been largely superseded by the use of IPG strips polyacrylamide gel strip to generate an for the following reasons: immobilized pH gradient. The pH gradients are created with sets of acrylamido buffers, which are  Carrier ampholyte tube gels must be cast by derivatives of acrylamide containing both reactive the user double bonds and buffering groups. The general  Carrier ampholyte – generated pH gradients structure is CH2=CH–CO–NH–R, where R drift over time and are, therefore, not as contains either a carboxyl [–COOH] or a tertiary reproducible as immobilized pH gradients amino group (for example, –N(CH3)2). These  Carrier ampholytes are complex chemical acrylamide derivatives are covalently incorporated mixtures, and batch-to-batch variations affect the into polyacrylamide gels at the time of casting and characteristics of the pH gradient can form almost any pH gradient (Righetti 1990).  Narrow pH gradients and gradients IPG strips are: encompassing the extremes of the pH range (below pH 4 and above pH 9) cannot be  Supplied commercially and ready to use accommodated  Prepared on a plastic backing to simplify  Tube gels can be difficult to handle handling  Highly reproducible and stable over even For More information on related products, please extended IEF runs (Bjellqvist et al. 1982) click: Aladdin  Available in a wide variety of pH gradients and lengths

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