Cell electrophoresis

The physical principle of cell electrophoresis, the role of media pH and ionic strength, and the nature of cell coat electric charge are considered. The advantages and defects of analytic and preparative cell electrophoresis variants are analyzed. The results of use of cell electrophoresis for studying and separation of erythrocyte, leukocyte and bone marrow cell suspension are presented.     

Reaction electrophoresis

The differential mobilities of compounds in an electric field are important analytical criteria and we can use them to bring electrophoretically pure components of a mixture medium on which they are separated. To this end, the compound undergoing reaction are brought into positions on the carrier to assure optimal contact between selected fractions, within a predetermined domain of time and distance. The appearance of a product defines their reactivities, and the product's continued migration on the same carrier can provide the first key to its identity as is demonstrated and discussed. The method is called reaction electrophoresis and it will be of particular use in studies with labile components. It is illustrated here with the coupling reaction of the sodium salt of 1,4-naphthol sulfonic acid and tetrazotized o-dianisidine.     

Capillary electrophoresis

While capillary electrophoresis, or historically related techniques, have been used for over a century, and recognition of the value of this separation methodology has certainly grown rapidly in the past few years, the technique has generally been used by analytical chemists, particularly in Europe and Japan, and small groups of researchers in the United States. Many of the basic instrumentation problems have been solved only relatively recently, and researchers using capillary electrophoresis are now turning their attention to studying specific applications which demonstrate the potential versatility of this electrophoretic technique. The appearance of standardized commercial instrumentation is imminent. With the availability of such technology, capillary electrophoresis will no longer be an academic curiosity, but rather a tool with the potential for routine separations of diverse samples of interest to analyst, researcher, and clinician.     

Three methods of capillary electrophoresis compared with high-resolution agarose gel electrophoresis for serum protein electrophoresis

We assessed the BioFocus 2000 capillary electrophoresis instrument for use in a routine clinical laboratory. We examined 210 serum samples received for serum protein electrophoresis by four methods: (1) The Bio-Rad HR015EC high-resolution serum protein kit on the BioFocus; (2) the Jenkins–Guerin (JG) method on the Applied Biosystems 270A HT Capillary Electrophoresis System (JG-ABI); (3) the Jenkins–Guerin method using the BioFocus (JG-BF); and (4) the quantitation of monoclonal bands found in 76 of the 210 samples was assayed by Helena Titan Hi-Res agarose gel electrophoresis (HRAGE). The correlation coefficient between the three sets of capillary electrophoresis monoclonal band results and the Helena quantitation was 0.92 or better. Although the quantitative comparison of monoclonal bands by HR015EC was very good, the lack of sharpness of monoclonal bands using the HR015EC kit meant our preference was to use the JG method on either the ABI or on the Biofocus.     

Capillary electrophoresis.

The past year has seen major advances in capillary electrophoresis in terms of broadening applicability. A variety of successful approaches to peptide/protein and DNA separation and analysis are now available, and techniques for saccharide analysis are developing rapidly. Capillary electrophoresis--mass spectrometry continues to demonstrate its potential as a tool for high-resolution structure analysis.     

Pulsed field electrophoresis

Pulsed field electrophoresis, or PFE, provides good separation between large molecules that under constant field electrophoresis are hard to isolate. This is due to the weak dependence of the constant field mobility on the molecular weight for these large molecules. If a spectrum of relaxation times exists that describes the recovery of the mobility to its constant field value after a reversal of the field, then we show that molecules with differing molecular weights are separated into two groups. Those with short relaxation times are unaffected by the cycling of the field and those with long relaxation times exhibit reduced mobilities. If the molecules adopt conformations that decrease their mobility initially, after a field reversal we demonstrate that a minimum develops in the mobility as a junction of the relaxation time. Using the model we demonstrate that effects of varying the switching times as a function of time. We predict that exponential rather than linear dependencies of the switching times on time increase the range of molecular weights over which enhanced separation can occur.     

Lectin affinity electrophoresis

Lectin affinity electrophoresis is a powerful technique to investigate the interaction between a lectin and its ligand. Affinity electrophoresis results from the reduced mobility of a charged species owing to its interaction with an immobile species. In this protocol, a two-dimensional lectin affinity electrophoresis experiment is described that affords separation of oligosaccharides. The first-dimension is composed of a weak, polyacrylamide, capillary tube gel containing a lectin. The example described involves a mixture of fluorescently labeled disaccharides. The mobility of only the lectin-binding disaccharide is reduced affording a separation in the first-dimension. The tube gel is then extruded and placed onto the second-dimension gradient polyacrylamide gel and subjected to electrophoresis. Mobility in the second-dimension is dependent on molecular size and visualization is by fluorescence under transillumination. This method is also applicable, with appropriate modifications, for the separation and analysis of glycopeptides and glycoproteins.     

Two-dimensional strandness-dependent electrophoresis

Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acids in complex samples according to strandness, conformation and length. Under the non-denaturing conditions of the first electrophoretic step, single-stranded DNA, double-stranded DNA and RNA.DNA hybrids of similar length migrate at different rates. The second electrophoretic step is performed under denaturing conditions (7 mol l(-1) urea, 55 degrees C) so that all the molecules are single-stranded and separate according to length only. 2D-SDE is useful for revealing important characteristics of complex nucleic acid samples in manipulations such as amplification, renaturation, cDNA synthesis and microarray hybridization. It can also be used to identify mispaired, nicked or damaged fragments in double-stranded DNA. The protocol takes approximately 2 h and requires only basic skills, equipment and reagents.