Bio-Rad Whole Gel Eluter and Mini Whole Gel Eluter - Manual (Page 17)

Section 6
SDS-PAGE
6.1 Reagents for SDS-PAGE Slab Gels (Laemmli buffer system) 3
Premixed liquid acrylamide (concentrated solution) and premixed electrophoresis buffers
are available (see product information section). Sections 6.2–6.4 describe preparation of poly-
acrylamide gels using the following stock solutions.
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.
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 (SDS-reducing buffer)
Deionized water 2.8 ml
0.5 M Tris-HCl, pH 6.8 1.0 ml
Glycerol 2.0 ml
10% (w/v) SDS* 1.6 ml
ß-mercaptoethanol 0.4 ml
0.5% (w/v) bromophenol blue 0.2 ml
Total Volume 8.0 ml
Mix equal volumes of sample buffer and sample, and heat at 95 °C for 4 minutes. SDS
reducing buffer is 0.06 M Tris-HCl, pH 6.8, 2% SDS, 5% beta-mercaptoethanol, 25% glyc-
erol (w/v) and 0.01% bromophenol blue.
*To make a 10% SDS solution: Dissolve 1.0 gram of SDS in deionized water with
gentle stirring, then adjust the volume to 10 ml with water.
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