When two strips have the same pH range but differ in length - the ratio of the strip lengths gives the relative focusing power of the longer strip.The calculations are performed as follows: The 7 cm pH 3–10 IPG strip is assigned a baseline focusing power of 1.0 to calculate the relative focusing powers of the other strips, as shown in the table above. Relative focusing power expresses the enhanced resolution expected in the first dimension when using IPG strips of different lengths or pH ranges. Bio-Rad's ReadyStrip IPG strips are produced using high-purity IPG monomers and are available in different lengths and in a variety of pH gradients The strip length required is dictated largely by the size of the second-dimension gels to be used, with longer strips and larger gels providing larger sample capacity and higher resolution. They are cast on plastic backings, making them much easier to handle than ampholyte gels. Commercially available IPG strips are easy to use and provide reproducible and stable gradients during extended IEF runs. These acrylamide derivatives are covalently incorporated into polyacrylamide gels at the time of casting and can form almost any conceivable pH gradient (Righetti 1990).Ī reproducible pH gradient is crucial for successful IEF. The general structure is CH2=CH–CO–NH–R, where R contains either a carboxyl or a tertiary amino group (e.g., –N(CH 3) 2). PH gradients for IPG strips are created with sets of acrylamido buffers, which are derivatives of acrylamide containing both reactive double bonds and buffering groups. IPG strips are much more difficult to cast than carrier ampholyte gels (Righetti 1983) however, IPG strips are commercially available (see ReadyStrip IPG Strips for available gradients and sizes). IPG strips offer the advantage of gradient stability over extended focusing runs (Bjellqvist et al. In IEF, a mixture of proteins is resolved on a pH 3–10 IPG strip according to each protein's pI and independently of its size.Ī stable, linear, and reproducible pH gradient is crucial to successful IEF. Moreover, in IEF, proteins migrate to their steady-state positions from anywhere in the system. By contrast, proteins in conventional electrophoresis continue to move through the medium until the electric field is removed. Proteins approach their respective pI values at differing rates, but remain relatively fixed at those pH values for extended periods. In this way, proteins condense, or are focused, into sharp bands in the pH gradient at their individual characteristic pI values.įocusing is a steady-state mechanism with regard to pH. If, on the other hand, it diffuses into a region of pH greater than its pI, the protein will become negatively charged and will be driven toward the anode. If this protein should happen to diffuse to a region of lower pH, it will become protonated and be forced back toward the cathode by the electric field. There, being uncharged, it will stop migrating (see figure below). Eventually, the protein will arrive at the point where the pH gradient is equal to its pI. As it migrates, its net charge and mobility will decrease and the protein will slow down. During migration through the pH gradient, the protein will either pick up or lose protons. When a protein is placed in a medium with a pH gradient and subjected to an electric field, it will initially move toward the electrode with the opposite charge. Related Topics: Second-Dimension Separation, Protein Sample Preparation for 2-D Electrophoresis, Staining and Visualization of Proteins After 2-D Electrophoresis, Imaging and Analysis of 2-D Electrophoresis Gels, Protein Spot Excision and Protein Identification, and Troubleshooting Electrophoresis Gels with 2-D Doctor™. This section provides technical details to perform successful IEF using IPG strips. IEF can be performed using two techniques: immobilized pH gradients (IPG) with ampholytes covalently bound to a gel, or carrier ampholytes that migrate through a gel to generate the pH gradient. In this way, each protein in a sample becomes "focused" according to its pI. When a protein reaches a pH value that matches its pI, its net electrical charge becomes neutral, and stops migrating. The proteins separate as they migrate through the pH gradient in response to the applied voltage. IEF works by applying an electric field to protein within a pH gradient. The first dimension in a 2-D gel electrophoresis experiment involves the separation of proteins according to their isoelectric point (pI) by isoelectric focusing (IEF).
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