Protein isoelectric focusing

Chapter 5 includes sufficient details on IEF to provide a better understanding of this technique. It also includes a number of applications of various proteins. Isoelectric focusing is applicable only to the fractionation of amphoteric species, such as proteins and peptides, that can act both as acids and bases. Nonamphoteric species, nucleic acids in particular, cannot be resolved by IEF. Both analytical and preparative modes of IEF, included in this chapter, have been developed as valuable tools for studying proteins. 

Two modifications of classical hemoglobin electrophoresis are also useful for studying these proteins isoelectric focusing and electrophoresis of individual globin chains, with or without the heme. These techniques can be used to identify some hemoglobins that are not separable by electrophoresis of the complete hemoglobin tetramers. 

The liver constitutively produces single-chain pro-MSP, which is released into the circulating blood. In contrast to the recombinant protein, isoelectric focusing studies show that the native material has secondary structure. The isoelectric point (pi) of the native protein in different samples of human serum was 5.5 to 6.2. In 6 M urea, the pi shifted to 7.6 (Leonard etal., 1982),... 

High-Resolution Two-Dimensional Gel Electrophoresis of Proteins Isoelectric Focusing and Nonequilibrium pH Gradient Electrophoresis (NEPHGE)... 

Isoelectric Focusing. Isoelectric focusing is a technique used for protein separation, by driving proteins to a pH where they have no mobiUty. Resolution depends on the slope of a pH gradient that can be achieved in a gel. 

Metallothionein (from rabbit liver) [9038-94-2], Purified by precipitation to give Zn- and Cd-containing protein fractions and running on a Sephadex G-75 column, then isoelectric focusing to give two protein peaks [Nordberg et al. Biochem J 126 491 1972]. 

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. 

Rossieretal. [332] usedUV excimer laser photoablation to cut channels 50 microns deep by 100 microns wide in laminated PET. These channels were filled with PA, and rapid separation of proteins by isoelectric focusing was demonstrated. 

Figure 4-5. Two-dimensional lEF-SDS-PAGE.The gel was stained with Coomassie blue. A crude bacterial extract was first subjected to isoelectric focusing (lEF) in a pH 3-10 gradient. The lEF gel was then placed horizontally on the top of an SDS gel, and the proteins then further resolved by SDS-PAGE. Notice the greatly improved resolution of distinct polypeptides relative to ordinary SDS-PAGE gel (Figure 4-4).

Manning JM et aJ Normal and abnormal protein subunit interactions in hemoglobins.] Biol Chem 1998 273 19359-Mario N, Baudin B, Giboudeau J Qualitative and quantitative analysis of hemoglobin variants by capillary isoelectric focusing. J Chromatogr B Biomed Sci Appl 1998 706 123-Reed W, Vichinsky EP New considerations in the treatment of sickle cell disease. Annu Rev Med 1998 49 46l. 

Isoelectric focusing of transferrin is a useful biochemical test for assisting in the diagnosis of these conditions truncation of the oligosaccharide chains of this protein alters its isolectric focusing pattern... 

Separation of the purified PL1, PL2 and PL3 isoenzymes by SDS-PAGE yielded single protein bands that corresponded to a molecular mass of 42 kDa (6), 4 kDa higher than calculated from the deduced amino acid sequences. Isoelectric focusing revealed a pi of >10 for each isoenzyme similar to that of the basic Ech PLs (15). 

SDS-PAGE revealed only one protein band in the purified AE fractions with a MW of 42,000 D (Fig.2). Isoelectric focusing of AE showed that pi > 9. The amino acid composition of the purified AE is shown in table 1. 

However, after the preparative isoelectric focusing column, the PNL was the only band detected both by lyase activity staining (B 3) and by protein staining (a 3). 

Figure 5. Analytical isoelectric focusing. Ultrathin layers (0.4 nun) of polyacrylamide with ampholytes pH 2-11 were used. Samples of 10 pg of protein in 10 pi of 1 % glycine were applied. A.- Silver staining. B.- Stain for activity on overlays containing pectin in tris/HCl buffer at pH 8.0 with CaClj M.- Broad pi Calibration Kit protein (Pharmacia), samples of 5 pg of protein were applied. 1.-Ammonium sulphate precipitated proteins from cultures on pectin. 2.- Fractions with PNL activity eluted from the Superdex 75HR1030 column. 3.- Purified PNL.

Evidence of pectic enzymes secreted by FORL species has been obtained (3). The total proteins of FORL subjected to isoelectric focusing were resolved into several bands widely distributed between pi 5.9 and 7.45. FORL (rj) presented one mayor band of activity with a pi of 7.0 however, FORL (r ) presented one characteristic and principal band at 7.45, slightly more basic than these reported by others authors (4). 

There are many proteins in the human body. A few hundreds of these compounds can be identified in urine. The qualitative determination of one or a series of proteins is performed by one of the electrophoresis techniques. Capillary electrophoresis can be automated and thus more quantified (Oda et al. 1997). Newer techniques also enable quantitative determination of proteins by gel electrophoresis (Wiedeman and Umbreit 1999). For quantitative determinations, the former method of decomposition into the constituent amino acids was followed by an automated spectropho-tometric measurement of the ninhydrin-amino add complex. Currently, a number of methods are available, induding spectrophotometry (Doumas and Peters 1997) and, most frequently, ELISAs. Small proteins can be detected by techniques such as electrophoresis, isoelectric focusing, and chromatography (Waller et al. 1989). These methods have the advantage of low detection limits. Sometimes, these methods have a lack of specifidty (cross-over reactions) and HPLC techniques are increasingly used to assess different proteins. The state-of-the-art of protein determination was mentioned by Walker (1996). 

Righetti, P. G., Isoelectric focusing of proteins in conventional and immobilized pH gradients, in Protein Structure A Practical Approach, Creighton, T. E., Ed., IRL Press, New York, 1989, 23. 

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