| Isoelectric focusing (IEF)
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| Figure 2.15 SDS-PAGE. Sodium dodecylsulfate-polyacrylamide gel electrophoresis is used to separate proteins on the basis of their molecular weights. Larger molecules are retarded in the gel matrix, whereas the smaller ones move more rapidly. Lane A contains standard proteins with known molecular masses (indicated in kDa on the left). Lanes B, C, D, and E show results of SDS-PAGE analysis of a protein at various stages in purification: B = total protein isolate; C = ammonium sulfate precipitate; D = fraction from gel permeation chromatography; E = purified protein from ion exchange chromatography. |
| Isoelectric focusing (IEF) is used for to separate proteins on the basis of their pI by conducting electrophoresis in a microchannel or gel containing a pH gradient. A protein applied to the system will be either positively or negatively charged, depending on its amino acid composition and the ambient pH. Upon application of a current, the protein will move towards either the anode or cathode until it encounters
that part of the system that corresponds to its pI, where the protein has no charge and will cease to migrate. IEF is used in conjunction with SDS-PAGE for two-dimensional gel electrophoresis (Figure 2.16). This technique is particularly useful for the fractionation of complex mixtures of proteins for proteomic analysis (below).
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