Electrophoresis techniques

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). 

Smithies vertical starch gel electrophoresis (S7) separates the plasma proteins more distinctly than any other method. If the Hp concentration is normal, the Hp type can generally be recognized directly after the staining for proteins, but sensitive and more specific staining for heme groups, e.g., benzidine, o-dianisidine (04), and malachite green (N5) are preferable. This technique consumes more hydrolyzed starch than the simpler original horizontal electrophoresis technique (S5). 

What is capillary electrophoresis and what advantages does it have over other conventional electrophoresis techniques. 

Capillary electrophoresis is an electrophoresis technique in which the mixture components are separated in a capillary tube and detected with an on-line detector after the separation occurs. The advantages include smaller quantity of sample and qualitative and quantitative analysis in a much shorter time. 

Electrophoresis is normally run in aqueous media, hence the analytes must be soluble in water. Presently only three types of water-soluble dendrimers have been successfully analyzed using gel electrophoresis techniques. The list includes Starburst PAMAM dendrimers [21], nucleic acid dendrimers [21] and poly(lysine) dendrimers [23, 24] (see Figures 10.2, 10.4 and 10.6). However, in each case appropriate water solubilizing terminal groups are required (i.e. -NH2, -OH or C02H groups) for suitable electrophoretic analysis. 

Sardas S, Aygun N, Gamli M, et al Use of alkaline comet assay (single cell gel electrophoresis technique) to detect DNA damage in lymphocytes of operating room personnel occupationally exposed to anaesthetic gases. Mutat Res 418(2-3) 93-100, 1998... 

With micelles, microemulsions, or liposomes, a second phase is introduced into the separating system. As in chromatography, exchange of the analyte between the mobile and the stationary phases controls the separation process. Contrary to classical chromatography, both phases are mobile, moving with different velocities. As in all electrophoresis techniques, the net mobility of an analyte is the mean mobility of its fraction in the aqueous and the micellar phases ... 

MECC has also been reported to be an effective method for the separation of enantiomers [269, 270]. Tsai et al. [271] reported y-cyclodextrin-modified micellar capillary electrophoresis technique for the separation of dansylated-DL-amino acids. 

Fig. 5.4.12a-d Electropherograms of pure standards containing nine BA conjugates at a concentration of 50 nmol/ml (a), a blank serum sample from a healthy subject (b), the same serum sample spiked with 50 nmol/ml of nine different BAs (c) and serum sample from a patient with chronic hepatitis infection (d) analyzed by the capillary electrophoresis technique. Ultraviolet absorbance detection at 195 nm (reprinted from [30])... 

To test this idea, a Bio-Rad Protean II electrophoresis cell and Bio-Rad Model 3000xi computer-controlled power supply were used to carry out the electrophoretic separation and recovery of pre-stained and unstained protein calibration standards (lysozyme, soybean trypsin inhibitor, carbonic anhydrase, ovalbumin, bovine serum albumin and phosphorylase B) obtained from Bio-Rad Laboratories. Standard SDS-gel electrophoresis techniques were used [167]. 

A. W. Moore-Jr, J. P. Larmann-Jr, A. V. Lemmo and J. W. Jorgenson, Two-dimensional liquid chromatography-capillary electrophoresis techniques for analysis of proteins and peptides , Methods Enzymol. 270 401-419 (1996). 

The application of electrophoresis techniques to the determination of anions in various types of water is reviewed in Table 13.1. 

Other modifications of capillary electrophoresis techniques, described below, have been adopted for the improvement of separation of several substances, including proteins, peptides and aminoacids. Furthermore, new procedures are also being developed to avoid the adsorption of proteins and peptides onto the walls of the capillary and consequently improving their selectivities. 

H. Chassaigne, C. C. Chery, G. Bordin, F. Vanhaecke, A. R. Rodriguez, Two-dimensional gel electrophoresis technique for yeast selenium-containing proteins. 

H. Chassaigne, C. C. Chery, G. Bordin, F. Vanhaecke, A. R. Rodriguez, 2-Dimensional gel electrophoresis technique for yeast selenium-containing proteins - sample preparation and MS approaches for processing 2-D gel protein spots, J. Anal. Atom. Spec-trom., 19 (2004), 85-95. 

Capillary sieving electrophoresis (CSE) — An electrophoresis technique, in which analytes are separated in a capillary, containing a sieving medium (e.g., an entangled polymer network) in the -> supporting (background) electrolyte. The separation is based on differences in size and shape of the charged analytes. 

Huang, Z., Timerbaev, A.R., Keppler, B.K., Hirokawa, T. Determination of cisplatin and its hydrolytic metabolite in human serum by capillary electrophoresis techniques. J. Chromatogr. A 1106, 75-79 (2006)... 

The electrophoresis technique most often used with proteins is SDS-PAGE, i.e., polyacrylamide gel electrophoresis carried out in the presence of 0.1 % sodium do-decyl sulphate (See and Jackowski 1989). This technique, which was developed by Shapiro, Vinuela and Maizels, separates proteins, or more accurately protein subunits, exclusively on the basis of their molar mass. SDS is an anionic detergent that binds to proteins up to a level of about 1.4 g g 1 of protein, and in so doing it disrupts the quaternary, tertiary and, to a large extent, the secondary structure of the... 

The electrophoresis techniques that we have described have three significant drawbacks ... 

Table 4-11. Capillary electrophoresis techniques and their applications...

Gel electrophoresis is such an important technique for protein and nucleic add analysis, that it was natural to try and develop capillary-based electrophoretic methods. Although this has been achieved in prindple, CGE has not yet supplanted the classical vertical or horizontal electrophoresis techniques. This is probably due to the fact that with classical techniques it is possible to analyse multiple samples simultaneously, and also that the equipment and techniques are relatively simple. In contrast, CE analyses are usually carried out sequentially, the equipment is much more expensive, and the experimental approach more involved. However, CGE has several important advantages, notably high throughput rate, direct quantitation, and ease of automation these advantages are... 

Despite its lower resolution compared to 2-DE, 2-DB can also be applied in combination with the difference gel electrophoresis technique (DIGE) (Urdu et al. 1997 Reinders et al. 2006b), enabling the highly reproducible differential analysis of biological membrane samples. 

Boutin, J. A., Hennig, P., Lambert, P. H., et al. (1996) Combinatorial peptide libraries Robotic synthesis and analysis by nuclear magnetic resonance, mass spectrometry, tandem mass spectrometry, and high-performance capillary electrophoresis techniques. Anal. Biochem. 234, 126-141. 

For further reading on the micro-electrophoresis technique, see the review by A.M. James, cited in sec. 4.11c. 

A.M. James, Electrophoresis of Particles in Suspension in Surface and Colloid Science, Vol. II (1979). R.J. Good. R. Stromberg. Eds.. Plenum (1979), 121. (Detailed diiscusslon of the micro-electrophoresis technique). 

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