Electrophoresis affinity

Various modifications of affinity electrophoresis (for review see Ref. 179) are based on electrophoresis of proteins or other macromolecules in gel media containing immobilized or quasi-immobilized ligands capable of specific interaction with the electrophoresed substance. 

Two major variations of the method are used at present. The first approach is closely related to some immunoelectrophoretic techniques, e.g., crossed Immunoelectrophoresis or rocket electrophoresis [180,181], the only difference is that usually a lectin instead of an antibody is incorporated in the agarose gel the conditions in the gel (electroendoosmosis and pH) are used such that the lectin has a very low electrophoretic mobility. The lectin reacts with specific glycoproteins and forms characteristic precipitation lines analogously to the reactions observed during Immunoelectrophoresis between the antibody and the antigen. This method can be 

Some suggested electrolyte systems for the isotachophoretic separation of amino acids and peptides 

Characterization of the buffer system Leading ion Counter ion Terminating ion Additions to the terminator Notes 

Neutral and acidic amino acids as anions Cr (5-10 mM) Ammediol (to pH 8.6-9.7) -Alanine Ba(OH)2 to pH 10 Sensitive to atmospheric CO2 

Usually a non-covalent immobilized reactant is incorporated into the gel and the electrophoretic conditions are chosen in that way that a significant motion of this reactant does not occur. 

Especially in the case of low molecular mass ligands these ligands are covalently attached to the gel matrix, e.g., by copolymerization of allylesters of mono- or oligosaccharides. 

A special case of affinity electrophoresis is immunoelectropho-resis (cf. Chap. 4.10), which analyzes antibody-antigen interactions. 

The ligand is dissolved in Soln. B with a concentration of 0.2-1 mg/ml (for a 10 x 10-cm plate 5 ml are needed). Heat this solution to 60 °C and mix with an equal volume of agarose solution A, also heated to 60 °C. Pour the mixture onto a horizontally straightened glass plate. Introduce the comb and allow the gel to solidify at room temperature. 

Mix the samples with Soln. B in a 1 1 ratio supplement some bromophenol blue. 

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The examples mentioned up to now utilized strong Ag-Ab interactions using the equilibrium-mixture mode of ACE. But the quantitation of weak antigen-antibody interaction is also possible by ACE, if the mobility-shift approach, sometimes also called dynamic equilibrium affinity electrophoresis, is applied. 

Mangru, S.D., Harrison, D.J., Chemiluminescence detection in integrated postseparation reactors for microchip-based capillary electrophoresis and affinity electrophoresis. Electrophoresis 1998, 19(13), 2301-2307. 

Fig. 6.4 Affinity electrophoresis of the recombinant phosphorylases from potato tuber.,3) A crude bacterial cell extract containing the type-L isozyme (lane 1), the chimeric enzyme (lane2), or the type-H isozyme (lane 3) was electrophoresed in 5% polyacrylamide gels supplemented with 0 (A), 50 (B), and 500 pg/ml (C) glycogen. After electrophoresis, the gels were stained for enzyme activity in KI-b solution. (Reproduced with permission from Goldsmith and Fletterick, Pure and Appl. Chern., 55(4), 583 (1983)).

Affinity chromatography, based on biological recognition, has become a major means for the purification of biologically active molecules 2). The technique provides a simple and effective way of purification. Specific adsorption of the enzyme to its competitive inhibitor attached to a polymer matrix is the basis for an efficient enzyme purification. Affinity electrophoresis is also based on biological recognition3 . 

Capillary affinity electrophoresis (CAE) or affinity capillary electrophoresis (ACE) — An electrophoretic separation technique (- electrophoresis), in which -> analytes are separated in a capillary, with the -> supporting (background) electrolyte containing substances capable of specific, often biospecific, interactions with the analytes. Ref [i] Riekkola ML, Jonsson jA, Smith RM (2004) Pure Appl Chem 76 443... 

Combination of electrophoresis in different formats with affinity reactions started in the early 1950s, when Tiselius cells were used for the first time to characterise antigen-antibody interactions. A real milestone report in the field was the determination of the equilibrium constants for the binding of Ca2+ and Zn2+ to serum albumin by gel electrophoresis in 1960 [1]. Finally, the term "affinity electrophoresis" was proposed [2,3]. Its modification or, better said, its particular case, named immunoelectrophoresis, has been used for many years in reports relying on agarose gel separation in conjunction with immunoprecipita-tion. Appearance of precipitated zones on a gel is indicative for the antigen-antibody reaction and can be used for the identification of an analyte and also for its quantification. 

Horejsi and Kocourek devised a new procedure for the detection of lectins which they termed 127 affinity electrophoresis. This technique... 

Guttman A, Cooke N (1991a) Capillary gel affinity electrophoresis of DNA fragments. Anal Chem 63 2038-2042. 

In principle, affinity electrophoresis is carried out in polyacrylamide gels. The receptor protein moves to a given position relative to a tracking dye or other protein. Addition of the macromolecular ligand in various concentrations in the gel retards the movement of the receptor protein. If in addition, a small haptenic molecular oligosaccharide is added to the gel, the mobility is restored. From either set of measurements the of the receptor-macromolecular ligand or receptor-hapten interaction can be calculated. 

J. Breborowicz and A. Mackiewicz, Eds., Affinity Electrophoresis Principles and Applications, CRC Press, Boca Raton, FL, 1991. 

Wheat germ agglutinin (WGA), a lectin, has been used to measure BAP by affinity chromatography, affinity electrophoresis, or differential precipitation of isoforms. 

Mattiazzo M, Ramasamy I. Wheat germ lectin affinity electrophoresis of serum alkaline phosphatase with commercially available gels. Clin Chem 1993 39 1404-7. 

B13. B0g-Hansen, T. C., Affinity electrophoresis. Quantification and characterisation of glycoproteins with lectins. In Electrophoresis 79 . (B. J. Radola, ed.), pp. 193-202. Walter de Gruyter, Berlin, 1980. 

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