Bio-Rad PROTEAN II XL Cell - Manual (Page 44)

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14.2 Separating Gel Preparation - 0.375 M Tris, pH 8.8
12% a 7.5% b
Distilled water 33.5 ml 48.5 ml
1.5 M Tris-HCl, pH 8.8 25.0 ml 25.0 ml
10% (w/v) SDS stock
(store at room temperature)
1.0 ml 1.0 ml
Acrylamide/Bis (30% stock)
(Degas for ≥15 minutes at room
temperature)
40.0 ml 25.0 ml
*10% ammonium persulfate
(fresh daily)
500 µl 500 µl (0.05%)
TEMED 50 µl 50 µl (0.05%)
TOTAL MONOMER+ 100 ml 100 ml
* To make the 10% ammonium persulfate solution, dissolve 100 mg APS in 1 ml dH
2
O.
+ One can prepare any desired volume of monomer solution by using multiples of the 100 ml recipes.
a. For SDS treated proteins in the approximate molecular weight range between 10 and 100 K Daltons.
Use Bio-Rad’s Low MW Standards (catalog number 161-0304) for 12% separating gel.
b. For SDS treated proteins in the approximate molecular weight range of 40 –250 K Daltons. Use in con-
junction with Bio-Rad High MW SDS-PAGE Standards (catalog number 161-0303).
Calculated Volumes* (in ml) Required Per Gel Slab
Spacer Thickness
16 cm Length 20 cm Length
0.5 mm 12.8 ml 16 ml
0.75 mm 19.2 ml 24 ml
1.0 mm 25.6 ml 32 ml
1.5 mm 38.4 ml 48 ml
3.0 mm 76.8 ml 96 ml
*Volume required to completely fill gel sandwich to top of plates. Amounts may be adjusted depending on
application (with or without comb, with or without stacking gel, etc.).
14.3 Stacking Gel Preparation - 4.0% gel, 0.125 M Tris, pH 6.8
16 cm 20 cm
Distilled water 6.1 ml 12.2 ml
0.5 M Tris-HCl, pH 6.8 2.5 ml 5 ml
10% (w/w) SDS 100 µl 200 µl
Acrylamide/Bis (30% stock) (Degas
for 15 minutes at room temperature)
1.3 ml 2.6 ml
10% ammonium persulfate
(fresh daily)
50 µl 100 µl (.05%)
TEMED 10 µl 20 µl (0.1%)
TOTAL STOCK MONOMER 10 ml* 20 ml**
*Enough for two 0.75 mm or 0.50 mm gels.
**Enough for two 1.5 or 1.0 mm gels.