Bio-Rad Model 491 Prep Cell and Mini Prep Cell - Manual (Page 43)

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Prepare Ornstein-Davis Acrylamide Gels
Use the following table to prepare acry lamide gels (both analytical and prepar ative). The amounts listed for the
components in the table below are based on a total vol ume of 10 ml. Determine the total vol ume needed and
multiply each component by the appro priate number.
Table 13. Reference table for the preparation of acrylamide gels (10 ml volume).
%T Deionized H
2
O,
ml
Gel Buffer*,
ml
Bis-acrylamide solution
30% stock (37.5:1), ml
4 6.15 2.50 1.33
5 5.80 2.50 1.67
6 5.55 2.50 2.00
7 5.15 2.50 2.33
8 4.80 2.50 2.67
9 4.45 2.50 3.00
10 4.15 2.50 3.33
*Resolving Gel buffer - 1.5 M Tris-HCl 8.8
*Stacking Gel buffer - 0.5 M Tris-HCl 6.8
Prior to pouring a gel, induce acrylamide polymerization by adding the follow ing catalyst solutions. Amounts
are per 10 ml gel volume.
Table 14. Reference table for the preparation catalyst solutions.
Catalyst
10% APS**
(µl)
TEMED**
(µl)
Analytical Resolving Gel 50 5
Stacking Gel 50 10
Preparative Resolving Gel 25 2.5
Stacking Gel 50 10
**Note: To slow the rate of polymerizaton and avoid temperature induced in consistencies in then gel, different amounts of catalyst are
used for preparative gels and analytical gels. To make 10% APS, dissolve 100 mg in 1 ml of deion ized water.
Consult Sections 3 and 4 in this manual for an in-depth discussion about casting preparative gels, sample
loading and running conditions.
Running Conditions for Discontinuous Native-PAGE Using the Prep Cell
A. Run the Model 491 prep cell at 12 W con stant power. Cooling the lower buffer and (or) running the
gel in the cold room will help to maintain the biological activity of some proteins.
B. An elution flow rate of 0.75–1.0 ml/minute is recommended. Fractions should be collected following
elution of the ion front.
C. Protein migration rates of 1.0–2.0 cm/hour correspond to ap proximate elu tion times of 3–6 hr for a
5 cm gel.
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