Analysis of Electrophoresis Results

By separating biochemicals on the basis of charge, size, and conformation, electrophoresis can provide valuable information, such as purity, identity, and molecular weight. Purity is indicated by the number of stained bands in the electropherogram. One band usually means that only one detectable component is present that is, the sample is homogeneous or electrophoretically 

The identity of unknown biomolecules can be confirmed by electrophoresis on the same gel, the unknown alongside known standards. This is similar to the identification of unknowns by gas chromatography and HPLC as discussed in Chapter 3. 

As previously discussed in this chapter, the molecular size of protein or nucleic acid samples may be determined by electrophoresis. This requires the preparation of standard curves of log molecular weight versus fi (mobility) using standard proteins or nucleic acids. 

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The presence of two new tumor antigens in human colon with adenocarcinoma is demonstrated by the results of the coupled analysis of electrophoresis and agar diffusion (6, 7, 8, and 9) of Fig. 37. Such antigens are absent in extracts of normal colon tissue (gel 5). The method is readily adaptable to analysis of tumors of other tissues and of other diseased tissue. 

Plaque samples were collected before and 7, 15, 30 min after a sucrose rinse for each individual. Lactic, acetic, butyric, formic, propionic, pyruvic and succinic acids were determined by capillary electrophoresis. By analysis of the results, only lactic acid showed a significant difference in the RC group compared with the other two groups (table 2). Of note, the elevated lactic acid concentration was sustained for longer in the RC group than in the other groups, possibly caused by the reduced salivary flow, and may be responsible for the serious caries. 

RT-PCR can be performed in a semi-quantitative manner [8, 23] for large numbers of genes. This requires a control mRNA, which is reverse-transcribed and amplified along with the sample. Analysis of the resulting PCR products resolved with polyacrylimide gel electrophoresis, involves calculating the ratio of the sample to the control band on the gel. In this way, a relative measure of gene expression can be obtained. 

The standard Rodbard-Ogston-Morris-Killander [326,327] model of electrophoresis which assumes that u alua = D nlDa is obtained only for special circumstances. See also Locke and Trinh [219] for further discussion of this relationship. With low electric fields the effective mobility equals the volume fraction. However, the dispersion coefficient reduces to the effective diffusion coefficient, as determined by Ryan et al. [337], which reduces to the volume fraction at low gel concentration but is not, in general, equal to the porosity for high gel concentrations. If no electrophoresis occurs, i.e., and Mp equal zero, the results reduce to the analysis of Nozad [264]. If the electrophoretic mobility is assumed to be much larger than the diffusion coefficients, the results reduce to that given by Locke and Carbonell [218]. 

High-efficiency separations of FQ-labeled proteins are only achieved in the presence of an anionic surfactant, such as SDS. As a result, capillary isoelectric focusing is not useful for the analysis of these proteins. Instead, we employ capillary sieving electrophoresis and micellar electrokinetic capillary chromatography for our two-dimensional electrophoresis. 

Capillary gel electrophoresis is becoming very widely used in the biotechnology field for the high resolution separation of DNA and peptides according to molecular weight, but it has limited application for the analysis of surfactants (Wallingford, 1996). CGE does result in an increase in the resolution per unit time over SEC for charged polymers (Poli and Schure,1992). 

When fresh or frozen tissue is used for proteomic analyses, the results cannot be related directly to the clinical course of diseases in a timely manner. Instead, researchers frequently reduce the number of interesting proteins to a manageable number and then attempt to use immunohistochemistry to understand the implications of proteomic changes in archival formalin-fixed, paraffin-embedded (FFPE) tissue for which the clinical course has been established.3 Unfortunately, immunohistochemistry is a semiquantitative pro-teomic method, and the choice of interesting proteins must occur without advance knowledge of the clinical course of the disease or the response to therapy. If routinely fixed and embedded archival tissues could be used for standard proteomic methods such as 2-D gel electrophoresis and mass spectrometry (MS), these powerful techniques could be used to both qualitatively and quantitatively analyze large numbers of tissues for which the clinical course has been established. However, analysis of archival FFPE tissues by... 

However, such an explicit evaluation of Qa is very difficult and one can only hope to evaluate it in very simple models moreover, it is clear from a simple dimensional analysis that the result (443) indeed gives the correct order of magnitude. We shall thus not dwell upon this point any further rather, after this rather detailed analysis of the velocity field, we shall now consider the problem of electrophoresis. 

In the version we used, electrophoresis of proteins makes it possible to separate proteins and evaluate molecular weight and relative amount of each fraction. Analysis of this data, therefore, has permitted us to detect the following types of modification of proteins resulting from photodynamic treatment ... 

We found that it is necessary to run several sets of differential display primers prior to an analysis of the distribution of differential display bands. This allows for a comparison between different independent reactions using different PCR primers to assess the quality of individual cDNA samples and discriminate between sample-to-sample variability and potential positive bands that are consistently found in different repUcates. The presence or absence of a specific band in lanes corresponding to independent experimental samples indicates a reproducible difference in the relative amount of cDNA in a given sample, which should reflect differences in mRNA levels. However, the interpretation of the differential display results is not always straightforward. For example, a thick band can reflect quantitative differences in the initial concentration of a specific cDNA between samples or can represent comigration of two bands. Replication of the PCR reactions for samples that have differences in banding pattern will eliminate a significant number of false positive differential display differences. Also, in some cases, it may be informative to alter the electrophoresis conditions to maximize resolution of a band of interest prior to isolation, reamplification, and further analysis of potential positive bands. 

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