Crossed electrophoresis

A modified technique consisting of an electric field applied perpendicular to a flowing buffer solution and using chromatography paper as support(cross-electrophoresis) has been exploited to study the reaction of neomycin with heparin 9 184. Evidence for the formation of a neomycin-heparin complex was obtained by this means. 

Laurell, C. B. (1965) Antigen anubody crossed electrophoresis. Anal Btochem. 10, 358-361. 

Weeke, B. (1970) Carbamylaied human transfemn used as a reference in the Laurell crossed electrophoresis. Scand.J. Clin Lab. Invest. 25, 161-163. 

Svendsen, P. J. and Axelsen, N. H. A. (1972) A modified antigen-antibody crossed electrophoresis characterizing the specificity and titre of human precipitins agamst Candxda albicans. J. ImmuncL Methods 1,169-176. 

Laurell CB. Antigen-antibody crossed electrophoresis. AnalBiochem 1965 10 358-61. 

B19. Bours, J., Zauzig, H. D., and Rink, M., The crystallin composition of bovine lens epithelium, equator and nucleus, in relation to aging A comparison of isotachophoresis, isoelectric focusing and antigen/antibody crossed electrophoresis. In Biochemical and Biological Applications of Isotachophoresis (A. Adam and C. Schots, eds.), pp. 207-220. ELsevier, Amsterdam, 1980. 

Many modifications have been made since the original technique was described, several of them permit quantitative evaluation. The most common of these methods are rocket electrophoresis, crossed electrophoresis, jused rocket electrophoresis, intermediate gel electrophoresis, and tandem electrophoresis. The basic arrangement for each is described below. 

Crossed electrophoresis with two currents acting perpendicularly was described by McDonald and Urbin [24]. The electrophoretic chamber has four electrodes, two of which are positive, two are negative. After the potential has been applied, the substances to be separated start to move along the diagonal. Therefore this technique is sometimes referred to as continuous diagonal electrophoresis. The technique was applied to the separation of DNP amino acids. 

The two-dimensional version of cross paper electrophoresis resembles two dimensional separations with a buffer change before the second run. This version is applicable when one or both reactants are not pure compounds. Crossed electrophoresis has found its wide applicability in immunochemical studies, in studies about enzyme-substrate interactions, and in interactions of proteins with a number of low molecular weight compounds. 

Nakamura, S. (1966) Cross Electrophoresis — Its Principle and Applications, pp. 28-38, Igaku Shoin, Tokyo and Elsevier, Amsterdam. 

See crossed electrophoresis, counter electrophoresis, immuno-electrophoresis... 

Techniques for the detection or assay of various substances based on the reaction of those substances with specific antibodies (or vice versa, i.e. the detection and assay of antibodies using antigens). Such techniques include agglutination reactions, automated immune precipitation, complement fixation tests, crossed electrophoresis, counter electrophoresis, double diffusion, enzyme immunoassay, fluoroimmunoassay, haem-agglutination, immunoelectrophoresis, immunofluoresence, radial immunodiffusion, spin immunoassay, immunofixation, immunoradiometric assay and radioimmunoassay. See separate entries for these subjects. 

Transferrin may be determined directly using immunochemical methods such as radial immunodiffusion or crossed electrophoresis. It can be measured indirectly by determining the amount of iron it can bind (see iron binding capacity). 

Schematic diagram showing a cross section of a capillary column for capillary electrophoresis.

Molded polyamide surfaces can be hardened by grafting with Ai,Ai-diallylacrylamide [3085-68-5] monomer under exposure to electron beam (159). AijAZ-DiaHyltartardiamide [58477-85-3] is a cross-linking agent for acrylamide reversible gels in electrophoresis. Such gels can be dissolved by a dilute periodic acid solution in order to recover protein fractions. 

The use of agarose as an electrophoretic method is widespread (32—35). An example of its use is in the evaluation and typing of DNA both in forensics (see Forensic chemistry) and to study heritable diseases (36). Agarose electrophoresis is combined with other analytical tools such as Southern blotting, polymerase chain reaction, and fluorescence. The advantages of agarose electrophoresis are that it requires no additives or cross-linkers for polymerization, it is not hazardous, low concentration gels are relatively sturdy, it is inexpensive, and it can be combined with many other analytical methods. 

Polyacrylamide Electrophoresis. Polyacrylamide gels are synthesized through the combination of acrylamide [79-60-1] (qv), CH2=CHC0NH2, monomer and a cross-linking comonomer (see Acrylamide POLYMERS). Typically, the cross-linking comonomer of choice is... 

N,]S2-diaHyltartardiamide (DATD) [58477-85-3] (37). The cross-linking of polymerized monomer with the comonomer is what controls the pore size of the gel polymer mesh. This level of pore size control makes polyacrylamide gel electrophoresis an effective analytical tool. 

Fig. 3. Immunological reactions, where Ag is antigen and Ab is antibody, for detection in electrophoresis (a) Ouchtedony technique (b) single-radial diffusion (c) rocket immunoelectrophoresis and (d) crossed immunoelectrophoresis.

Very recently, the Shipman group have made a further step towards a comprehensive structure/activity profile for noncovalent interactions between azinomycin B and DNA [152]. They synthesized simplified azinomycin analogues 69 and 96-98 (Scheme 11.13), retaining both the epoxide and aziridine alkylating functionalities, with systematically altered substitution on the naphthoate fragment, and analyzed their DNA crosslinking by gel electrophoresis. They found that cross-... 

Small-angle X-ray scattering (SAXS), circular dichroism (CD), and UV spectroscopy at different temperatures were used to investigate the nature of calf-thymus DNA in aqueous solution, in the presence of [Me Sn] " (n = 1-3) species. The results demonstrate that the [MeSn(IV)] moiety does not influence the structure and conformation of the DNA double helix, and does not degrade DNA, as indicated by agarose gel electrophoresis. Inter alia, the radii of gyration, Rg, of the cross section of native calf-thymus DNA, determined by SAXS in aqueous solution in the presence of [Me Sn] " (n = 1-3) species are constant and independent of the nature and concentration of the [Me Sn] species. 

The structure of these gel-like systems of micelles is very different from that of conventional electrophoresis media made from chemically and physically cross-linked polymers of polyacrylamide and agarose [75], The absence of chemical or physical cross-links in the Pluronic gel-like phases may allow a larger degree of freedom for macromolecular transport around the obstacles that make up the medium than occurs in conventional electrophoresis media. 

Boyd, BM Prausnitz, JM Blanch, HW, High-Frequency Altemating-Cross-Field Gel Electrophoresis with Neutral or Shghtly Charged Interpenetrating Networks to Improve DNA Separation, Electrophoresis 19, 3137, 1998. 

Chrambach, A Rodbard, D, Polyacrylamide Gel Electrophoresis, Science 172, 440, 1971. Chu, B Yeh, F Sokolov, EL Starodoubtsev, SG Khokhlov, AR, Interaction of Slightly Cross-linked Gels of Poly(diallyldimethylammonium chloride) with Surfactants, Macromolecules 28, 8447, 1995. 

Gelfi, C Righetti, PG, Polymerization Kinetics of Polyacrylamide Gels I. Effect of Different Cross-Linkers, Electrophoresis 2, 213, 1981. 

Theory Cross-flow-electrofiltration can theoretically be treated as if it were cross-flow filtration with superimposed electrical effects. These electrical effects include electroosmosis in the filter medium and cake and electrophoresis of the particles in the slurry. The addition of the applied electric field can, nowever, result in some qualitative differences in permeate-flux-parameter dependences. 

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). 

FIGURE 9.6 The loss of cross-linking activity of an aqueous solution of QMP11 (100 pM) in the presence (black) and absence (gray) of dA (20 mM). Cross-linking activity was measured at the indicated times by addition of duplex DNA (3 pM) and subsequent analysis by denaturing gel electrophoresis. Source Adapted from Angew. Chem. Int. Ed. 2008, 47, 1291-1293.69... 

In the previously described electrophoretic methods, the capillary was filled with electrolytes only. Another mode of operation in capillary electrophoresis involves filling the capillary with gel or viscous polymer solutions. If desired, a column can be packed with particles and equipped with a frit.68 This mode of analysis has been favorably used for the size determination of biologically important polymers, such as DNA, proteins, and polysaccharides. The most frequently used polymers in capillary gel electrophoresis are cross-linked or linear polyacrylamide,69 cellulose derivatives,70-75 agarose,76 78 and polyethylene glycols. 

Originally, polyacrylamide was used as an anticonvective additive in slab gel electrophoresis,79 but later its molecular sieving capability was also utilized.80 Polyacrylamide is a polymer built exclusively from monomeric units, with or without cross linking.81 A chemically cross-linked network is... 

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