Bio-Rad Model 491 Prep Cell and Mini Prep Cell - Manual (Page 39)

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Continuous Buffer Systems
The pH attained in the resolving gel of the Ornstein-Davis system ap proaches pH 9.5, which may be outside
the range of stability for some pro teins. Alternative dis continuous buffer systems derived for preparative work
can be found in an arti cle by Chrambach and Jovin (Chrambach and Jovin 1984). The electrophore sis
buffers described in this article span the pH range from 3–10. Protocols for using these discon tinuous buffers
are analagous to those listed in Section 3 for the Ornstein-Davis buffer system.
If discontinuous systems cannot be used because of stacking-induced ag grega tion, a continuous buffer
system will be required. In continuous systems the same buffer is used in the upper and lower elec trode
chambers and in the gel. McLellan describes various continu ous buffer systems from pH 3.8–10.2 that can
be tried (Mclellan 1982). See Section 9.4 for preparation of these continu ous buffers.
For in-depth discussions of electrophoresis and the distinctions between contin uous and discontinuous
systems, the Hames, Andrews, and Allen references in Section 9.5.
Optimizing Conditions for Preparative Native-PAGE
Conditions for purification of native proteins with the Model 491 prep cell should first be optimized on a small
scale using mini-slab gels. It is recommended that the proce dures described in this guide be repeated for
each new sample to be purified. Any op timization proce dure should be carried out using the same crude or
partially purified protein sample as will be ap plied to the Model 491 prep cell. Figure 16 presents an overview
of the optimization procedure.
Isolating individual proteins by preparative native-PAGE can be simplified by par tially purifying the sample
using preparative isoelectric focusing or chromatogra phy before electrophoresis in the Model 491 prep cell.
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