Bio-Rad Model 491 Prep Cell and Mini Prep Cell - Manual (Page 29)

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Fig. 13. Elution profile and SDS-PAGE analysis. A, Elution profile. Chromatographically purified phycocyanin was separated into
three sub units (18.5 kD, 21 kD, and 23 kD) by preparative SDS-PAGE in the Model 491 prep cell. B, Aliquots from the Model 491 prep
cell fractions 9-32 containing the separated protein subunits of phyco cyanin were analyzed by SDS-PAGE gels (14%T) and silver
stained. Starting material was run in the extreme left and right lanes. The low molecular weight subunit (18.5 kD) eluted in the Model
491 prep cell fractions 9–17, the mid-sized subunit (21 kD) in fractions 18–29 and the largest subunit (23 kD) eluted in fractions 29–32.
Example 2:
Purification of a 98 kD Subunit from a 96 kD Subunit of Keyhole Limpet Hemocyanin
Keyhole limpet hemocyanin analyzed on SDS-PAGE gels shows several bands cov ering a broad range of
molecular weights. See Figure 15A. Among these are two proteins of approximately 96 kD and 98 kD
representing a differ ence in molecular weight of approximately 2%. To determine the optimal %T for
resolving these two proteins, four analytical gels of 5%, 6%, 8%, and 10%T/2.67%C were cast.
Hemocyanin was electrophoresed on each gel along with two lanes of prestained SDS-PAGE standards.
The prestained standards were used to monitor the position of the proteins of interest. Runs were con tinued
until the prestained BSA marker (~80 kD) of the prestained standards had just run off the gel. The gels were
silver stained and dried, and the dis tance between the two hemocyanin bands (96 kD and 98 kD) was mea-
sured and plotted versus %T. An optimal %T of approximately 7% is indi cated for resolving these two
proteins. See Figure 14.
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A
1 hr 2 hr 3 hr 4 hr 5 hr
Ionic
contaminants
10 20 30
18.5 kd
21 kd
23 kd
kD
23
21
18.5
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
B
kD
kD
kD