Isoelectric focusing typing

The more abundant lyase produced by FORL has been purified to homogeneity as it is shown by analytical isoelectric focusing (figure 5). The data in Table 1 show that a 32.48-fold increase in specific activity is achieved with a recovery of approximately 2.36%. The enzyme showed an "endo" type of action and a great specificity for pectin. 

Three major types of isoelectric focusing first dimensions are used currently (S7) ... 

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...

Zone electrophoresis is influenced by adsorption and capillarity, as well as by electroosmosis. Therefore evaluation of mobility (and f) from this type of measurement is considerably more complex than from either microelectrophoresis or moving-boundary electrophoresis. Nevertheless, zone electrophoresis is an important technique that is widely used in biochemistry and clinical chemistry. One particularly important area of application is the field of immunoelectrophoresis, which is described briefly in Section 12.11. Additional information on zone electrophoresis may be obtained from Probstein (1994) and Hunter (1981) and the references given there. Variants of zone electrophoresis also exist see, for example, Gordon et al. (1988) for information on a variant known as capillary zone electrophoresis and Righetti (1983) for information on what is known as isoelectric focusing. 

The two most widely used types of electrophoresis tests are (i) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and (ii) isoelectric focusing (IEF). 

The isoforms of apoE were first clearly demonstrated by Utermann et al., who showed on one-dimensional isoelectric focusing of VLDL apolipoproteins that there were four major isoforms of apoE (U3— U5). These were named, from acidic to basic, apoE-1, apoE-2, apoE-3, and apoE-4. Patients with Type I1 hyperlipoproteinemia (dysbetalipoproteinemia) were, in virtually all cases, deficient in apoE-3 (U3-U6). Only 27% of subjects had apoeE-4, but this band was not associated with any particular abnormality. However, individuals deficient in apoE-3 were also deficient in apoE-4 (U6). Utermann suggested that the genetic pattern of apoE isoforms could be explained if there was a single genetic locus for apoE which could produce three apoE phenotypes, i.e., apoE-N (normal), apoE-D (deficient, associated particularly with the Type III disorder), and apoE-ND (U4, U6). 

W4. Wamick, G. R., Mayfield, C., Albers, J. J., and Hazzard, W. R., Gel isoelectric focusing method for specific diagnosis of familial hyperlipoproteinemia type 3. Clin Chem. 25, 279-284 (1979). 

When transversely oriented focusing forces of the type used in isoelectric focusing or isopycnic sedimentation are used to confine different components to different thin bands or laminae in an FFF channel, we have a variant of FFF called hyperlayer FFF. Suppose component A is focused at y = 15.2 /xm and B at y = 127 /xm (0.005 in). You may assume that the thin bands form a 6-function distribution in coordinate y and that channel thickness w = 254 /xm. What is the R value for each of the two components ... 

Capillary isoelectric focusing separations can be discussed from two major viewpoints, the capillary type and the detection techniques used, but other practical questions, e.g., ampholytes, sample pretreatment, etc., should also be considered. 

The most distinguishing phenomenon in capillary electrophoresis is whether the experiments are performed in the absence or in the presence of electroos-motic flow (EOF), (see Chapter 6 for details on EOF). Unlike other types of capillary electrophoresis, isoelectric focusing can be performed under both modes. Since the experimental and theoretical principles governing these modes of CIEF are different, they will be discussed separately. 

Capillary isoelectric focusing will be applied mostly for proteome studies in the future. Proteome analysis requires fast methods with high separation efficiencies in order to screen the various cell and tissue types for their proteome expression and monitor the effect of environmental conditions and time on this expression. The established two-dimensional gel electrophoresis is by far too slow for a... 

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