Bio-Rad Whole Gel Eluter and Mini Whole Gel Eluter - Manual (Page 21)

C. Stacking Gel Buffer Stock
0.5 M Tris-HCl, pH 6.8
Dissolve 6 g Tris base in approximately 60 ml deionized water.
Adjust to pH 6.8 with 1 N HCl (Do not back titrate).
Make to 100 ml with deionized water and store at 4 °C.
D. Sample Buffer
Deionized water 4.8 ml
0.5 M Tris-HCl, pH 6.8 1.0 ml
Glycerol 2.0 ml
0.5% (w/v)bromophenol blue 0.2 ml
Total Volume 8.0 ml
E. 10x Electrode (Running) Buffer, pH 8.3 (Makes 1 Liter)
Tris base 30.3 g
Glycine 144.0 g
Dissolve in deionized water and adjust the final volume to 1,000 ml. DO NOT adjust pH
with acid or base. To make 1 liter of 1x electrophoresis buffer (0.025 M Tris, 0.192 M glycine,
pH 8.3) dilute 100 ml of 10x stock with 900 ml deionized water.
7.3 Prepare Ornstein-Davis Acrylamide Gels
Use the following table to prepare gels with %T ranging from 4%T to 10%T.
% T Deionized Gel buffer Acrylamide/Bis solution
(acrylamide H
2
O solution* 30% stock (37.5:1)
monomer) (ml) (ml) (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% 4.47 2.50 3.00
10% 4.17 2.50 3.33
* Resolving Gel buffer - 1.5 M Tris-HCl, pH 8.8
* Stacking Gel buffer - 0.5 M Tris-HCl, pH 6.8
Catalysts 10% APS* TEMED*
Resolving Gel 50 µl 5 µl
Stacking Gel 50 µl 10 µl
* Amounts are per 10 ml gel volume. To make 10% APS, dissolve 100 mg in 1 ml of deionized water. TEMED is used neat.
7.4 Reagents for Continuous Native-PAGE
In continuous systems, the same buffer is used in the electrode chambers and in the gels.
Since stacking gels are not commonly employed, proteins migrate in bands at least as wide as
the applied sample. Therefore, the sample volume must be kept at a minimum, i.e., the sam-
ple should be at as high a concentration as possible. The mobilities of proteins in continuous
systems are dictated primarily by pH rather than by sieving through the polyacrylamide gel.
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