Bio-Rad Model 491 Prep Cell and Mini Prep Cell - Manual (Page 46)

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Prepare Resolving Gels
As protein mobilities are best modified by pH, continuous nondenaturing PAGE systems use relatively large
pore size gels. Generally 4–6 cm long gels are suffi cient for optimum resolution in the prep cell.
For 10 ml acrylamide monomer solution
Table 18. Acrylamide monomer solution per 10ml of gel volume.
%T Deionized H
2
O,
ml
Continuous Buffer*,
ml
Acrylamide/bis solution
30% stock (37.5:1), ml
4 6.65 2.00 1.33
5 6.30 2.00 1.67
6 5.85 2.00 2.00
*In continuous systems the same buffer is used in the upper and lower elec trode chambers and in the gels.
Prior to pouring a gel, induce acrylamide polymerization by adding the follow ing catalyst solutions. Amounts
are per 10 ml gel volume. To cast a prepara tive gel refer to Sections 3 and 4 in this manual.
Table 19. Catalyst quantity recommendation by type of gel.
Catalyst
10% APS**
(µl)
TEMED**
(µl)
Analytical Resolving Gel 5 5
Stacking Gel 50 10
Preparative Resolving Gel 25 2.5
Stacking Gel 50 10
**Note: Below pH 6, TEMED becomes less effective as a catalyst. Between pH 4 and pH 6, increasing the concentration of TEMED
5-fold to 10-fold will poly merize the gel.
Sample Preparation
Sample buffer for continuous native-PAGE is a dilution of the electrophore sis buffer. Tracking dyes are
generally not used. The concentration of the sample buffer is generally 1/10 that of the running buffer.
Glycerol is added to the sample buffer to a final concentration of 20%.
Important: Keep the protein sample as concentrated as possible since there are no stacking gels used with
these buffer systems. Bands will be at least as wide as the sam ple that is loaded onto the preparative gel.
Running Conditions for the Model 491 Prep Cell
For continuous buffer systems, run the gels at 5 W constant power (approximately 10 mA and 500 volts).
Cooling the lower buffer and (or) run ning the Model 491 prep cell in the cold room may help to maintain the
biological ac tivity of some proteins. Refer to Section 3.6 in this manual for de tails about cooling the
apparatus.
The elution buffer flow rate for the Model 491 prep cell should be set to 0.75–1.0 ml/min. Native proteins
migrate at individual velocities dependent on the pH of the buffer system used. Therefore, it is difficult to
predict exactly in which frac tions the protein of interest will elute. An enzyme assay or im munoblot can be
used to identify the specific location of the protein in a slab-gel or in the fractions collected from the prep
cell. Analysis by SDS-PAGE can be used to confirm the resolution and purity.
For a complete discussion regarding operating the Model 491 prep cell and details about sample collection
and analysis, refer to Sections 3 and 4.
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