Bio-Rad Whole Gel Eluter and Mini Whole Gel Eluter - Manual (Page 20)

Section 7
Native-PAGE
7.1 Native PAGE Slab Gels
The discontinuous buffer system of Ornstein-Davis (Tris/chloride/glycine) should be the
first non-denaturing gel system tried. 4 An advantage of discontinuous systems for dilute pro-
tein solution is the use of stacking gels to concentrate the sample. However, the stacking phe-
nomena encountered in discontinuous systems can cause aggregation of some proteins and this
can severely interfere with resolution.
The pH attained in the resolving gel of the Ornstein-Davis system approaches pH 9.5,
which may be outside the range of stability for some proteins. Alternative discontinuous
buffer systems derived for preparative work can be found in an article by Chrambach and
Jovin.
5 The electrophoresis buffers described in this article span the pH range from pH 3 to
pH 10.
If discontinuous systems cannot be used because of stacking-induced aggregation, a con-
tinuous buffer system will be required. In continuous systems the same buffer is used in the
upper and lower electrode chambers and in the gel. McLellan describes various continuous
buffer systems from pH 3.8–10.2 which can be tried.
2 See Section 7.4 for preparation of these
continuous buffers.
7.2 Reagents for Discontinuous Native PAGE (Ornstein-Davis)
A. 30% Acrylamide Stock Solution
Acrylamide/Bis (30% T, 2.67% C)
146 g acrylamide (29.2 g/100 ml)
4 g N ' N ' -Bis-methylene-acrylamide (0.8 g/100 ml)
Dissolve in about 350 ml deionized water.
Make to 500 ml with distilled water. Filter and store at 4 °C in the dark.
Or substitute Bio-Rad’s Preweighed Acrylamide/Bis 37.5:1 mixture or 40%
Acrylamide/Bis Stock solution.
Caution: Acrylamide monomer is a neurotoxin. Avoid breathing acrylamide dust, do
not pipet acrylamide solutions by mouth, and wear gloves when handling acrylamide
powder or solutions containing it. For disposal of unused acrylamide, add bis-acrylamide
(if none is present), induce polymerization, and discard the solidified gel.
B. Separating (resolving) Gel Buffer Stock
1.5 M Tris-HCl, pH 8.8
Dissolve 54.5 g Tris base in approximately 150 ml deionized water.
Adjust to pH 8.8 with 1 N HCl (Do not back titrate).
Make to 300 ml with deionized water and store at 4 °C.
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