Isoelectric focussing

SDS-PAGE was performed by the method of Laemmli [17]. The methods for native PAGE, isoelectric focussing, detection of esterase activity in electrophoresis gels, and assays for protein glycosylation have been described elsewhere [5]. 

Subsequently four different CE modes are described in the sections Capillary Zone Electrophoresis, Capillary Gel Electrophoresis, Capillary Isoelectric Focussing, and Micellar Electrokinetic Chromatography (MEKC), respectively. The fundamental principles of the specific separation modes are briefly explained, using appropriate equations where required. In Table 3 all equations are listed. In addition, the influence of both instrumental parameters and electrolytic solution parameters on the optimization of separations is described. 

Electrophoresis is one of many electromigrational separation techniques which include isotachophoresis, immunoelectrophoresis and isoelectric focussing that have been used to separate various species on the basis of their different mobility in an electric field. These techniques can be used not only to achieve separations but also it is possible to identify the ligand bound to the metal. This can be done by comparing the isoelectric points, immunological behaviours, extent of mobilities or step heights of the sample constituents with those of well-characterised standards. A difficulty, however, is in the determination of the metal constituent itself. Except in the case of radioisotopes, the activities of which can be easily measured, non-radioactive elements can be detected only after further separation steps. 

Metallothionein (from rabbit liver) [73767-16-5]. Purified by precipitation to give Zn- and Cd-containing protein fractions and running on a Sephadex G-75 column, then isoelectric focussing to give two protein peaks [Nordberg et al. BJ 126 491 7972]. 

The third example concerns an industrially relevant enzyme, glucose isomerase (see Chapters 7,10, and 19). Table 8.6 reproduces the purification data, and Figure 8.10 shows the SDS PAGE and IEF (isoelectric focussing) diagrams. 

Righetti, P.G., Brown, R. R and Stone, A. L. (1978). Aggregation of ampholine on heparin and other acidic polysaccharides in isoelectric focussing. Biochim. Biophys. Acta 542, 232-244. 

Stenman, W. H., Characterisation of R-type vitamin B 12-binding proteins by isoelectric focussing. III. Cobalophilin (R protein) in myeloproliferative states and leucocytosis. Scand. ]. Clin. Lab. Invest. 35, 157-161 (1975). 

During 2DE, protein extracts undergo a combination of two different types of separation. First, proteins are resolved on the basis of their isoelectric points (pi) by isoelectric focussing (IEF). Second, focused proteins are resolved on the basis of their molecular weight (MW) by electrophoresis on a polyacrylamide gel (SDS-PAGE). Since any protein as well as its modifications are characterised by a particular pI/MW... 

Proteins can be separated according to their isoelectric point in a process known as -> isoelectric focussing. See also -> zwitter ion. 

P.G. Righettl. Isoelectric Focussing. Theory, Methodology and Applications. Elsevier (1983). 

The energy dependence of import into mitochondria has been exploited to accumulate large amounts of precursors in an uncoupler-poisoned living cell [93]. Precursor to the 8-subunit of the proton ATPase has been purified from such cells by affinity chromatography on an antibody column, followed by chromatofocussing and isoelectric focussing. After renaturation, this precursor can be correctly processed by the matrix protease and can be imported into mitochondria, but only in the presence of a proteinaceous factor from the yeast cytoplasm [94]. A similar finding has been reported for import of precursors into rat liver mitochondria in which a factor is provided by the reticulocyte lysate [95,96]. 

Zhou and Johnston [55] reported protein characterization by capillary isoelectric focussing (CIEF) on-hne coupled to RPLC-MS. Direct coupling of CIEF to ESl-MS is limited by interferences by the ampholytes. Inserting RPLC in-between can help removing these interferences. CIEF is performed in combination with a microdialysis membrane-based cathodic cell to remove the ampholyte and to collect protein fractions by stop-and-go CIEF prior to transfer to a 5><0.3-pm-ID C,8 trapping colunm and RPLC separation on a 50><0.3-pm-ID C4 column. The separation is performed using an acetonitrile-water gradient (0.1% acetic acid). ESI-MS is performed on a quadrupole-TOF hybrid (Q-TOF) instrument. 

One- and two-dimensional gel electrophoresis (ID- or 2D-GE) is an important tool in the separation and isolation of intact proteins [9], In ID-GE, the proteins are separated in a sodium dodecylsulfate poly(acrylamide) gel (SDS-PAGE). The separation is according to molecular weight. In 2D-GE, the proteins are first separated by isoelectric point (pi, isoelectric focussing, lEF), and next by molecular weight. 2D-GE is considered to be the most powerful tool in protein separation. Nevertheless, the technique suffers from problems it is labour-intensive, analysis time is long, and the reproducibility poor. Furthermore, hydrophobic proteins do not behave well in the first lEF step and tend to form broad bands. 

Fractionation by capillary isoelectric focussing (CIEF) prior to LC-MS was reported by several groups (Ch. 17.5.6). Retention time from the RPLC and pl-data from the CIEF separation were applied to validate potentially identified peptides from the SEQUEST search [27]. The initial number of 7629 identified peptides was rednced to 1837 identified and 1130 likely hits by pi and retention time validation. 

Keywords Capillary electrophoresis Capillary gel electrophoresis Capillary isoelectric focussing Polyelectrolytes Polystyrenesulfonates Polyvinylpyridines Polycarboxybetaines Polyampholytes... 

Fig. 30 Capillary isoelectric focussing of polyampholytes PA1-PA4. Separation conditions capillary PVA 40/50 cm, i.d. 75 pm BGE 300 ppm sample in MilliQ+2% Servalyt anolyte H3P04, catholyte NaOH /=+30.0 kV focussing time 12 min mobilization 30 kV+0.1 psi T=25 °C detection at 280 nm...

I See Ref. 13. [Reprinted, by permission, from R G. Righetti, Isoelectric Focussing Theory, Methodology and Applications", Elsevier, New York, 1983. 1983, Elsevier Science Publishers B.V.]... 

E. G. Richards and R. Lecanidou, in Electrophoresis and Isoelectric Focussing in Polyacrylamide Gels, R. C. Allen and H. R. Maurer, Eds., Walter de Gruyter, Berlin, 1974,... 

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