Ampholine, 457 also

In spite of the fact that chromatofocusing originated [45] from the formation of internal pH-gradient in a column of a conventional ion exchanger using conventional buffers, for its further development [46-49] not only were special ion exchangers synthesized (cf., section 4.5.2.4), but special amphoteric buffers were also applied. LKB Ampholines were used for experiments with isoelectric elution [47,48], but in the last paper of this series [49] Pharmacia Pharmalyte buffers (pH... 

Many experimenters blot the sought-after protein from the SDS gel onto a membrane that can be put directly into the sequencing machine after the blot buffer s glycine is removed. From the lEF gel, you can also blot (for example) with protein mixtures whose components do not differ in size. Nevertheless, before blotting the lEF gel the ampholines must be washed out with perchloric acid (Hsieh et al. 1988). Some protein gets lost, and sensitive bindings (e.g., Asp-Pro) are partially hydrolyzed due to the acidic pH. 

Capillary zone electrophoresis is not only the simplest form of CE, but also the most commonly utilized. Discussion of this mode permits the presentation of a generic design for the instrumentation for CE. The addition of specialized reagents to the separation buffer readily allows the same instrumentation to be used with the other modes mentioned in the previous section addition of surfactants with MEKC, ampholines for CIEF and a sieving matrix (linear polymers, entangled matrices) for CGE. The discussion on CZE in the following subsections allows for analysis of some of the basic principles governing analyte separation by this technique. 

Ampholine carrier electrolytes are supplied in the form of water solutions, 40% w/w, each covering a pi range of 2 pH units. In the near future also 1 pH unit ranges will be available. In order to get a more suitable distribution of the field strength, it is preferable to add a small amount, say, 10% of the total amount of carrier ampholytes of a batch with pH 6-8. This is especially important when working with extreme pH ranges. 

Ampholine carrier electrolytes cover the pH range 3-10. However, there are many ampholytes with high molecular weights and with isoelectric points under 3. There are also several biologically important substances with isoelectric points over 10 (cytochromes, histones, etc.). 

Staining the zones after focusing is somewhat of a headache. This is because practically all applicable protein dyes also stain the carrier ampholytes. Awdeh and co-workers have described a method of staining the proteins directly, even in presence of Ampholine chemicals. Otherwise it is necessary to fix the proteins and wash out the carrier amphol3rtes before staining can be done. Another way of removing the Ampholine before staining is by means of electrophoresis, as done by Dale and Latner (34). 

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. 

The electrophoresis seems to serve two main purposes. It spreads out the components which have been separated with high resolution. The components are then easier to study. The electrophoresis also helps to wash out the Ampholine electrolyte. 

Figure 34. Zone convection electrofocumi of a 40% Ampholine solution, pH 4-6. The electrofocusing time was 5 days and the voltage 800-1200 V. The contents of two different compartments were taken and diluted to 0.5%. They were electrofocused by density gradient electrofocuaing method. Each fraction covered about 0.5 pH units. pH-gradients with specified shapes can also be constructed by adding or removing different fractions (Valmet, 53).

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