Riley and Coleman

The mixtures are carefully layered above each other in each tube, the sample mixture being added to the middle layer. Polymerization can be done either by fluorescent light or by ammonium persulfate. The focusing was done with electrophoresis equipment from Canal Industries, Bethesda, Md. Phosphoric acid 1% was used as the anolyte, and 2% ethylene-diamine solution as the catholyte. After 4-6 hr, the focusing 

Another method is to fix and dye the gel at the same time, using 0.2% Fast Green for 1-2 hr. Clearing can then be done electrolytically. However, it is preferable to clear by soaking in the clearing bath overnight. 

The gel consists of 1% agarose (Mann) in 2% aqueous solution of Ampholine carrier ampholytes with a suitable pH range. 

The agarose solution is warmed slightly until it flows easily. It is then poured onto a normal microscope slide until it forms a layer 2 mm thick. When it has hardened, a small pit is made in the middle to receive the sample. Two grooves 1 mm wide are cut, parallel to the length of the slide, one groove on each side of the sample pit. Standard immunoelectropho-resis grade gel was used, for example, from Gelman, Shandon, etc. 

Riley and Coleman (30) have also presented work on immunoelectrofocusing. They used 1.5% agarose ( Seakem, Bausch and Lomb) on a microscope slide in the traditional way, except that the conventional buffers were replaced by 2% Ampholine solution, pH range 3-10. The anolyte and catholyte were the same as for gel electrofocusing in the polyacrylamide system, that is, 1% phosphoric acid and 2% ethylene diamine. Riley and Coleman did their immunoelectrofocusing on human serum. With agargel a certain degree of electroendosmosis is obtained which can influence the result. 

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Hitherto, gel electrofocusing has been carried out in thin layers and troughs, or else in narrow tubes. Very thin layers have been used by Awdeh et cU. (39), as well as by Catsimpoolas (28). Leaback and Rutter (37) used a special trough for casting slabs of polyacrylamide. The latter method permits a certain amount of preparative work. Riley and Coleman (30) also used thin layers. 

Several writers have described gel immunoelectrofocusing. Catsim-poolas (66) reported good results using agarose gels. Riley and Coleman (30) have also reported this application. 

The technique begins with gel electrofocusing in tubes, in a similar way to the method of Wrigley (29) or Riley and Coleman (30). After focusing, the gel is removed from the tube and applied to a thin layer gel. 

There are a number of different probes available, including detection probes, which are labelled with a fluorescence marker or, for example, digoxigenin for a further linkage with an antibody-enzyme complex and then a later colorimetric reaction with a chromogenic substrate (such as nitro blue tetrazolium for alkaline phophatase) (Helentjaris McCreery, 1996 Kempf et al., 2000), Capture probes are used to bind the target sequence (RNA or DNA) to a plate or another surface. In most cases the probes are labelled with biotin to react with avidin, which is coated on a plate (Riley, Marshall, Coleman, 1986). 

Riley, R. R, and M. K. Coleman. 1970. Isolation of C-reactive protein of man, monkey, rabbit and dog by affinity chromatography on phosphorylated cellulose. Clinica ChimicaActa 30 483-486. 

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