Polyacrylamide gels carrier ampholyte isoelectric

For many years rehydratable granulated dextran (Sephadex) or polyacrylamide (Bio-Gel) gels have been used for gel chromatography and in the form of hydrated gel layers for analytical and preparative isoelectric focusing (l t, 15). Recently, rehydratable layers of granulated gels on a plastic film have been developed ( ). The presence of carrier ampholytes or addition of 1-2 % glycerol was necessary... 

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. 

Isotachophoresis of proteins can also be done in dilute polyacrylamide gel, in which sieving effects are oppressed. The results are comparable with isoelectric focusing or disc electrophoresis. The isotachophoretic profile is analogous to the isoelectric focusing pattern this is because proteins are separated mainly according to their p7 values. Chrambach [138] has shown that the advantage of isotachophoresis over isoelectric focusing is that, in isotachophoresis, no precipitation of proteins takes place. Carrier ampholytes were applied by Svendsen [128] to separate protein zone. 

Figure 21. Isoelectric focusing in 5% polyacrylamide gdi 2% Ampholine carrier ampholytes, pH range 3.0-10.0. The left run represents laccase. The two runs to the right represent carhonic anhydrase B. Both samples were supposed to be very pure. Bromphenol blue staining according to Awdeh without fixation of proteins and without washing gel free from Ampholine. (Awdeh, Bruges conference 1969.)...

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