Bio-Rad Model 491 Prep Cell and Mini Prep Cell - Manual (Page 27)

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4.7 Examples of the SDS-PAGE Optimization Procedure
The following two examples demonstrate optimization protocols for separat ing two closely spaced proteins
on the Model 491 prep cell. In each example, a series of mini-slab gels were run to determine the best
acrylamide concentration to use in the preparative gel.
Example 1:
Purification of the Subunits of Phycocyanin
Phycocyanin, purified by ion exchange chromatography, contains two natu rally col ored blue protein subunits
of ~18.5 kD and ~21 kD and a third, uncol ored, 23 kD sub unit. In this case, since the subunits of
phycocyanin are well separated in size, it is possible to purify all three in a single run.
Five analytical mini slab gels, 12%, 14%, 16%, 18%, and 20%T/2.67%C, were cast to determine the %T
that would provide maximal separation of the two largest sub units. The migration of the blue protein bands
could be monitored directly without the use of prestained SDS-PAGE standards. Gels were run under
standard SDS-PAGE conditions until the faster running protein (18.5 kD) was 5 mm from the bot tom of the
gels. The gels were silver stained and dried, and the dis tances between the two blue phycocyanin bands
were mea sured and plotted versus %T. An optimal %T of approximately 14% for re solving these two
proteins was indicated by the breakpoint, or cusp, of this curve. Refer to Figure 11.
Fig. 11. Analytical SDS-PAGE for determining optimum %T for purifica tion of 18.5 kD and 21 kD phycocyanin protein subunit
bands.The break point of the curve is con sidered the monomer concentration for optimal separation. In this case a 14% monomer
concen tration is indicated.
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Phycocyanin: optimal %T for ~ 18,500 Da and ~ 21,000 Da
Distance between pr
otein bands, mm
%T