Bio-Rad Model 491 Prep Cell and Mini Prep Cell - Manual (Page 28)

23
To confirm the procedure, the Model 491 prep cell was run using the same %T range as the analytical gels.
Preparative run conditions are shown in Table 6.
Table 6. Preparative run conditions.
Gel Composition 12%–17% T/2.67% bis
Gel Height 5.5 cm
Gel Size 25 mm ID
Sample Load 1 mg total protein
Running Conditions 40 mA constant current (~250–350 V)
Running Time ~5 hr
For each run the resolution (R = peak separation/average peak width) was de ter mined. The results of the
preparative runs shown in Figure 12 show how the op timal %T produces maximum resolution. The same
optimum %T of 14% as with the mini gel method was found. This plot demonstrates loss of resolution at
non-optimal gel concentrations. Reconstruction experiments with addition of irrelevant proteins showed
that regardless of the complexity of the sample of which phycocyanin was a part, an optimal %T of 14%
best resolved its two largest protein subunits.
Fig. 12. Confirmation of optimum %T for preparative SDS-PAGE to sepa rate two phycocyanin protein subunits (~18.5 kD and
~21 kD). As in chromatog raphy, resolu tion of two adjacent compo nents is defined as the ratio of peak separation to the mean peak
width as suming the peaks are Gaussian in shape (i.e., R=peak separation/average peak width).
23
17 16 15 14 13 12 11
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
%T
Resolution
Preparative runs of phycocyanin - resolution vs. %T
Preparative runs of phycocyanin — resolution vs. %T
Resolution
%T