Bio-Rad Model 491 Prep Cell and Mini Prep Cell - Manual (Page 44)

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D. The migration rates of proteins run in Ornstein-Davis gels at 12 W con stant power will approximate those
shown in Table 15.
Table 15. Approximate migration rates of proteins run in Ornstein-Davis gels at 12 W constant power.
Rf Migration Rate
1.0 30 mn/cm gel
0.8 40 mn/cm gel
0.6 50 mn/cm gel
0.45 60 mn/cm gel
Rf values for specific proteins are obtained from the mini-gels that were run to optimize conditions for the
Model 491 prep cell. To calculate Rf values for specific proteins use this formula:
Distance that the protein of interest migrated
Distance that the tracking dye migrated
The Rf value obtained from a mini gel can be used to estimate when a protein will elute from the Model 491
prep cell when the same concentra tion of acrylamide is used in both the mini gel and the preparative gel. Rf
values below 0.45 may result in excessive band broadening and loss of resolution.
9.4 Continuous Native-PAGE
Selection of Continuous Buffer Systems
In continuous systems the same buffer is used in the elec trode chambers and in the gels. Since stacking
gels are not com monly em ployed, proteins mi grate in bands at least as tall as the ap plied sam ple. Therefore,
the sample volume must be kept at a minimum. The mobilities of proteins in continuous systems are
dictated primarily by pH rather than by sieving through the poly acrylamide gel. For this reason, 6%
polyacry lamide gels are recom mended for most appli cations. For very large proteins 4% or 5% gels may be
used.
1. Use Table 16 to prepare electrophoresis buffers within the operating pH range of the protein under
investi gation. Make sure the acidic and basic components used are compatible with the protein under
in vestiga tion.
2. It is difficult to predict the migration rate of pro teins in native buffer sys tems without preliminary analysis.
For a chosen buffer system, determine if the protein of interest is neg a tively or posi tively charged.
a. If the pI of the protein of interest is < pH of the buffer system, the protein is negatively charged and
will migrate to the anode.
b. If the pI of the protein of interest is > pH of the buffer system, the protein is positively charged and
will migrate towards the cathode. In this case make sure to reverse the elec trodes to the power
supply to ensure migration into the gel.
3. Prepare 6%T/2.67%C mini-gels with the selected buffers. See Section 9.3 for preparation of a 6%
acrylamide gel. Electrophoresis is carried out at ap proximately 200 V for about one hour.
4. Mini-gels should be silver stained to determine the level of contamination of the protein of interest. The
buffer system of the gel showing the best reso lu tion and exhibiting a migration rate for the protein of
in terest of approximately 0.5 mm/minute is ideal for the preparative gel.
Buffers used for continuous systems are not limited to those listed in the fol low ing section. Virtually any
buffer can be used if it proves appropri ate for the pro tein un der inves tigation.
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Rf =