Isoelectric focusing separation power

Two-dimensional gel electrophoresis (2DE) is a two-dimensional technique for protein separation, which combines isoelectric focusing and sodium dodecyl sulphate (SDS) electrophoresis. The high resolving power results from separation according to charge (isoelectric point) in the first dimension and size (mobility in a porous gel) in the second dimension. Depending on the gel size, from several hundred to more than 5,000 proteins can be separated. 

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. 

Figure 4. Preparative isoelectric focusing in a granulated gel of a desalted lyophilized culture filtrate (50 mg protein) from the late exponential growth phase of Thermoactinomyces sp. Separation carried out for 48 hr at a constant power of 8 W (29). (C>) CM-cellulase activity (A) Avicelase activity (6).

Figure 5. Analytical isoelectric focusing in the pH range 3.5-9.5 (Am-pholine PAG Plate, LKB-Produkter AB, Bromma, Sweden) of filtrate samples from a culture of Thermoactinomyces sp. Separation carried out for 1.5 hr at a constant power of 1 W/cm (30). Protein concentration 0.5 mg mL 1 50 pJL applied with glass filter paper (Whatman GF/A) (6).

Capillary isoelectric focusing (CIEF) is one of the separation techniques with the highest resolution power. Since the first experiments performed by Hjerten and co-workers in the mid-1980s [1-3], hundreds of papers have appeared about methodological aspects and utilization. 

Another test of the resolving power of this new technique is to compare such separations with those achieved with conventional procedures. Glasel (II) found that neurophysins I and II, which were derived in an apparoitly chromatographically pure form by isoelectric focusing, were... 

Electrophoretic techniques, such as two-dimensional electrophoresis or isoelectric focusing, lend themselves under appropriate conditions to the separation with excellent resolution of small amounts of samples. They have mainly found use as powerful methods of analysis since their general applicability in the areas of peptide purification and isolation has, until recently, been severely restricted by limitations in sample capacity and instrumental design. 

Isoelectric focusing and SDS gel electrophoresis can be combined in two-dimensional gel electrophoresis. The proteins are first separated by isoelectric focusing and then the tube or lane of the gel is placed along the top of a slab of SDS gel and electrophoresed again. This is a very powerful separation method. 

Electrophoretic modes include zone, gel sieving, isoelectric focusing, and isotachophoresis. But electrophoresis has a distinct advantage over HPLC for analysis of biopolymers a vastly superior resolving power, especially in a two-dimensional format, where two separation mechanisms can be used in succession. 

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