Isoelectric focusing, IEF

Innovations in separation science continued on this theme and provided one of the most powerful separation techniques used in biochemistry, where proteins are separated with isoelectric focusing (IEF) applied in one direction, and gel electrophoresis (GE) applied at aright angle to the first separation direction (O Farrell, 1975 Celis and Bravo, 1984). In this case, proteins are first separated according to their isoelectric point, measured in p/units, and then according to their molecular weight by gel electrophoresis. The size separation step is usually aided by addition of a surfactant, most typically sodium dodecyl sulfate (SDS), and the gel material is a polyacrylamide formulation. 

The major advantage of 2D-PAGE is that it enables simultaneous separation of thousands of unknown proteins, first by charge using isoelectric focusing (IEF) and... 

RNase A is a basic protein with an isoelectric point (pi) of 9.45 and a net positive charge in neutral solution.35 However, the conversion of positively charged lysine side chains to polar, but neutrally charged, methylol adducts would be expected to lower the pi of formalin-treated RNase A. To explore this further, RNase A was treated with 5% formalin and analyzed by isoelectric focusing (IEF) gel electrophoresis. Figure 15.5a shows that the pi values were shifted into the pH 6.0-7.4 range. Figure 15.5b shows the results of IEF... 

CE is a family of techniques similar to those found in conventional electrophoresis zone electrophoresis, displacement electrophoresis, isoelectric focusing (IEF), and sieving separations. Other modes of operation unique to CE include micellar electrokinetic chromatography (MEKC) and capillary electrochromatography (CEC). 

In 1956, Smithies and Poulik first used 2-DE combining paper and starch gel electrophoresis to separate serum proteins. Nearly 20 years later, polyacrylamide was applied as a support medium. Charge-based protein separation followed as isoelectric focusing (IEF), applied to SDS-PAGE. Later, urea and nonionic detergents were used in IEF-2DE. The most significant achievement was the separation of proteins from E. coli. 

Fig. 4.5.2 Actual strategies for CDG diagnosis. Initial investigations on CDG patients are routinely carried out by isoelectric focusing (IEF) of serum transferrin. With a CDG type I pattern, subsequent analysis should imply determination of phosphomannomutase (PMM) and phos-phomannose isomerase (PMI) activities. Further studies, like analysis of the lipid-linked- and protein-bound-oligosaccharides, determination of enzyme or sugar transporter activities and molecular biology studies often have to be performed in more specialised laboratories. HPLC High-performance liquid chromatography, TLC thin-layer chromatography...

Another important and effective use of electrophoresis for the analysis of proteins is isoelectric focusing (IEF), which examines electrophoretic mobility as a function of pH. The net charge on a protein is pH dependent. Proteins below their isoelectric pH (pHj, or the pH at which they have zero net charge) are positively charged and migrate in a medium of fixed pH toward the negatively charged cathode. At a pH above its isoelectric point, a... 

For isoelectric focusing (IEF) and SDS-PAGE gels, materials are those descnbed by O Farrell (1,2) and Laemmli (3). It should be noted that for IEF, acrylamide and Zus-acrylamide must be of the highest level of purity, and urea must be ultrapure (enzyme grade) (see Methods in Molecular Biology, Volume 3, Chapters 15-21). 

Polyacrylamide gel electrophoresis is one of the most commonly used electrophoretic methods. Analytical uses of this technique center around protein characterization, for example, purity, size, or molecular weight, and composition of a protein. Polyacrylamide gels can be used in both reduced and nonrcduced systems as well as in combination with discontinuous and isoelectric focusing (ief) systems. 

Figure B3.1.3 An isoelectric focusing (IEF) gel, pH 3 to 10. Lane 1, 4 pg purified egg white cystatin. Lane M, broad-range pi standards trypsinogen (pi 9.3), lentil lectin-basic band (pi 8.65), lentil lectin-middle band (pi 8.45), lentil lectin-acidic band (pi 8.15), myoglobin-basic band (pi 7.35 visible as a broad band), myoglobin-acidic band (pi 6.85), human carbonic anhydrase B (pi 6.55), bovine carbonic anhydrase (pi 5.85), a-lactoglobulin A (pi 5.20), soybean trypsin inhibitor (pi 4.55), and amyloglucosidase (pi 3.50) in order shown from top of gel. The pi values of the two purified egg white cystatin isomers were determined to be 6.6 (upper band) and 5.8 (lower band). Adapted from Akpinar (1998) with permission from author.

The key to the first dimension, isoelectric focusing (IEF), is sample preparation, as IEF... 

Fig. 17.6. Isoelectric focusing diagrammatic view of isoelectric focusing (IEF) of a re-hydrated linear immobilized pH 3-10 gradient strip (Biorad, UK).

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