Preparative electrophoresis in polyacrylamide gel

This category of separations is commonly carried in gel columns that are located between two electrode vessels. The elution chamber is positioned at the lower end of the column. The apparatuses are provided with either a cooling jacket or the column 

Spacer gel is prepared from the above solutions by mixing them in a ratio 

Lower (anodic) electrode buffer (pH 8.1) (identical with the elution buffer) HCl (1 M) 60.0 ml 

Elution buffer is identical with this but diluted with water 1 8 

Continuous elution requires rather complex equipment, and dilution of zones, sometimes quite considerable, cannot be avoided. Therefore discontinuous elution systems are also in use. As an example of intermittent elution the apparatus of 

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Total tRNA and 2% tRNA from yeast and bull s liver were obtained according to a previously described procedme [5]. An enriched fraction of specific tRNA was obtained by preparative electrophoresis in polyacrylamide gel [6]. The enriched tRNA preparations contained from 75 to 150 nmoles of tRNA. 

The great analytical power of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) makes it one of the most effective tools of protein chemistry and molecular biology. In the past, there have been many attempts to convert the technique from analytical to preparative scale, because by SDS-PAGE, one can resolve more than 100 protein species in 5—6 h. The number of papers that describe preparative elution from polyacrylamide gels is immense (for example, see refs. 1—5). In spite of the numerous variations m the procedure of elution, none of the available methods is entirely satisfactory. Some of the methods are very laborious, and others lead to loss of resolution or poor recovery. 

SDS Electrophoresis and Gel Scanning. Samples were prepared for SDS-polyacrylamide gel electrophoresis by a lysis-toiling procedure. 2 mL of a culture sample was lysed by sonication with a Branson sonicator and microtip at setting 4 40% duty for 4 minutes. 200 microliters of the sonicate was mixed with 100 microliters of SDS loading buffer (0.3 g SDS, 0.01 g bromophenol blue, 3 mL glycerol, 1.5 mL beta-mercaptoethanol, and 1.88 mL tris-HCl(pH 6.8) per 10 mL). The mixture was toiled for 5 minutes in a microfuge tube then 20-50 microliters were loaded into a lane. The samples were separated on a 10% SDS gel with a 2% stacking buffer (18) for 5 hours at 30 milliamps. 

Contamination Problems. As analytical techniques become more sensitive, contamination becomes more of a problem. For example, Ochs (25) reported that many erroneous bands in polyacrylamide gels run with SDS were from proteins found on the skin of the researchers handling the gels. Her work indicates the need to exercise extreme care in sample preparation. In addition, suitable sample blanks handled in a parallel fashion would enable the researcher to identify contamination problems. Secondary checking of materials by two-dimensional electrophoresis would also help identify the nature and type of contamination. 

The world of electromigration separations is sharply divided into two areas. Zone electrophoresis on paper and related procedures have (in spite of their wide applicability to diverse organic compounds) already passed their period of favour. The other branch is represented by the more recent techniques some of which have already became widely accepted (such as isoelectric focusing or separations in polyacrylamide gel) and the others that are at the moment in the centre of a rapid development like displacement electrophoresis (isotachophoresis). This chapter is devoted mainly to analytical procedures such as these which are governing the area of electromigration separations at the moment with a single exception flow deviation (curtain) electrophoresis which will be discussed in more detail because it offers several new dimensions in the separation field. The other preparative procedures are summarized only briefly. 

Isoelectric focusing in polyacrylamide gel today appears to be nearly the most popular version of this technique [142-145]. The essential components for the gel preparation are identical with those used with conventional polyacrylamide gel electrophoresis (see pg. 428). The only difference is that carrier ampholytes (2% w/w of the total gel volume) are incorporated into the gel solution before polymerization. The protein sample (free of salts) can be mixed with the sample gel solution or it can be loaded in the conventional way in a sucrose solution. In the latter case it is covered with a protective layer of ampholyte. 

A great variety of methods have been designed to extract acidic proteins from the chromosomes. They are all rather drastic, and it is not certain whether they allow the functional properties of the acidic proteins to survive the procedure. In one method designed in Bonner s laboratory [77] chromatin is prepared and then extracted with acid to exclude histones. After histone extraction, the residue is treated with sodium dodecyl sulfate. The DNA sediments and the proteins are collected in the supernatant. Ammonium sulfate precipitates the proteins, which can then be submitted to polyacrylamide gel electrophoresis. The polyacrylamide gel electrophoresis of such a preparation yields a multiband pattern. The patterns are similar for acid proteins extracted from rat kidney and liver, but different for acid protein extracted from pea bud chromatin and rat liver chromatin. 

Analytical Disc Electrophoresis. The FSH preparation subjected to polyacrylamide gel electrophoresis according to the method of Davies (1964) and Ornstein (1964) at pH 9.1 and l C showed two bands, as illustrated in Fig. 4. 

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