Sample application focusing

Tapered plates, prepared with a gradual increase in thiclcness of the layer from 0.3 nm to 1.7 am, can be used to improve resolution of the sample [215]. On the tapered layer the solvent front velocity decreases as the thickness of the layer increases. This results in the formation of a negative velocity gradient in the direction of solvent migration. As a result the lower portion of a zone moves faster than the top portion, keeping each component focused as a narrow band. Plates with concentrating zones are useful for optimizing sample application. 

Use a suitable apparatus connected with a recirculating temperature-controlled water bath set at 10°C and gels for isoelectric focusing with a pH gradient of 3.5-9.5. Operate the apparatus in accordance with the manufacturer s instructions. Use as the anode solution phosphoric acid R (98 g/1 H3PO4) and as the cathode solution 1 M sodium hydroxide. Samples are applied to the gel by filter papers. Place sample application filters on the gel close to the cathode. 

There are no fixed rules regarding the positioning of the sample on the gel. In general, samples should not be applied to areas where they are expected to focus. To protect the proteins from exposure to extreme pH, the samples should not be applied closer than 1 cm from either electrode. Preforming the pH gradient before sample application will also limit the exposure of proteins to pH extremes. 

Figure 4 Schematic representation of the sample application and the single-step capillary isoelectric focusing with electroosmotic zone displacement. A pressure application may standardize the migration of the focused zones toward the detection point.

Sample application can be done either by pressure or vacuum, but not electrophoretically as in other capillary electrophoretic systems. The length of the sample plug in experiments performed in the presence of EOF must be carefully determined. Long sample zones may result a focusing step that will not be completed before the moving pH gradient reaches the detection point... 

Many applications focus on the isotopic analysis of elements in environmental samples (water, rocks, soil, minerals), meteorites, biological tissues, and nuclear materials (such as nuclear fuels). A relatively new application has been the use of TIMS in nuclear safeguards and arms control treaty verification (IAEA, 2003). Of most interest to this discussion is the high quality isotopic analysis of actinide elements, especially U and Pu. Typical performance parameters are given in Table 17.6 for isotopic analysis of actinide elements and additional examples are given in Section 17.8. 

Figure 22. Apparatus for isoelectric focusing in thin layer polyacrylamide gels according to Awdeh, Williamson, and Askonas (7). (o) polyacrylamide gel 1 mm thick (b) glass plate (c) carbon electrodes 20 cm apart (d) site of sample application. Each sample, 100/400 Mg of protein in less than 50 m1, was pipetted onto the surface of the gel and spread over a rectangular area about 1X2 cm. (Awdeh el al, 39.)...

FIGURE 3 Horizontal IPG-Dalt of mouse liver proteins. First dimension IFF with IPG 4-9 separation distance 18 cm sample application anode. Focusing time 35,000 V hr. Second dimension horizontal SDS-PAGE, 12-15% T. Silver stain. A, actin Alb, albumin C, cytochrome b, Ml, M2, M3, mitochondrial proteins S, carbamyl phosphate synthetase T, tubulin (Reprinted with permission from Gorg, 1993). 

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