Bio-Rad PROTEAN II XL Cell - Manual (Page 43)

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Section 14
Appendix
14.1 Reagents and Gel Preparation for SDS-PAGE Slab Gels (Laemmli buffer
system)
Stock Solutions
A. 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)
Make to 500 ml with distilled water. Filter and store at 4 °C in the dark (30 days maximum).
Or substitute Bio-Rad’s Preweighed Acrylamide/Bis
37.5:1 mixture
(Catalog No. 161-0112, 30 g)
(Catalog No. 161-0106, 200 g)
150 g Acrylamide/Bis (30 g/100 ml) to 500 ml with dH
2
O
B. 1.5 M Tris-HCl, pH 8.8
54.45 g Tris base (18.15 g/100 ml)
~150 ml distilled water
Adjust to pH 8.8 with 1 N HCl. Make to 300 ml with distilled water and store at 4° C.
C. 0.5 M Tris-HCl, pH 6.8
6 g Tris base
~ 60 ml distilled water
Adjust to pH 6.8 with 1 N HCl. Make to 100 ml with distilled water and store at 4° C.
D. 10% SDS
Dissolve 10 g SDS in water with gentle stirring and bring to 100 ml with dH
2
O.
E. Sample Buffer (SDS reducing buffer) (store at room temperature)
Distilled water 3.8 ml
0.5 M Tris-HCl, pH 6.8 1.0 ml
Glycerol .80 ml
10% (w/v) SDS 1.6 ml
2 b-mercaptoethanol 0.4 ml
0.05% (w/v) bromophenol blue 0.4 ml
8.0 ml
Dilute the sample at least 1:4 with sample buffer, and heat at 95 °C for 4 minutes.
F. 5X Electrode (Running) Buffer, pH 8.3 (enough for 10 runs)
Tris base 45 g (15 g/l)
Glycine 216 g (72 g/l)
SDS 15 g (5 g/l)
to 3 liters with dH
2
O
Store at 4 °C. Warm to 37 °C before use if precipitation occurs.
Dilute 300 ml 5X stock with 1,200 ml dH
2
O for one electrophoresis run.
Do not adjust the pH with acid or base.