Bio-Rad PROTEAN® Plus Hinged Spacer Plates and Combs - Manual (Page 13)

F. Sample Buffer (SDS reducing buffer: 62.5 mM Tris-HCl, pH 6.8, 10% glycerol,
2% SDS, 5% b-mercaptoethanol)
Distilled water 4.0 ml
0.5 M Tris-HCl pH 6.8 1.0 ml
Glycerol 0.8 ml
10% SDS 1.6 ml
β-mercaptoethanol 0.4 ml
0.05% (w/v) bromophenol blue (in water) 0.2 ml
8.0 ml
Dilute the sample at least 1:4 with sample buffer. Heat at 95 °C for 4 minutes.
* Laemmli, U. K., Nature, 227, 680 (1970).
G. 5x Electrode (Running) Buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3)
Tris base 45.0 g
Glycine 216.0 g
SDS 15.0 G
Distilled water in 3 L. Do not adjust the pH with acid or base. Store at 4 °C. Warm to
37 °C before use if precipitation occurs. Dilute 300 ml of 5x stock with 1.2 L distilled water
to make 1.5 L 1x buffer.
8.2. Calculating Gel Volumes
The total gel volume required is described by the equation:
VT = (W) (L) (T) (N)
VT = Total gel volume; W = Gel width (cm); L = Gel length (cm); T = Gel
thickness (cm); N = Number of gel slabs required
Table 8.2 Calculated volumes a required per gel for the
PROTEAN II xi and Mini-PROTEAN II cells
PROTEAN II Mini-PROTEAN II
Gel Volume b GelVolume b
Spacer 16 cm 20 cm 7 cm
Thickness length length length
0.50 mm 12.8 ml 16.0 ml 2.9 ml
0.75 mm 19.2 ml 24.0 ml 4.4 ml
1.00 mm 25.6 ml 32.0 ml 5.8 ml
1.50 mm 38.4 ml 48.0 ml 8.8 ml
3.00 mm 76.8 ml 96.0 ml
Note: With a multi-gel casting chamber, the total gel volume required depends on several
factors, including gel thickness, number of gels, gel length, and amount of space-filling.
To make a precise determination of the volume required for a particular application, set
up the casting chamber with the glass plates, spacers, and combs to form the required
number of gel sandwiches and measure the volume of deionized distilled water required
to fill it to the desired level. Then disassemble the chamber and rinse and dry the parts.
a. The volumes reported completely fill the gel sandwich to the top of the inner plate. The
amount of separating gel may be adjusted depending on application (with or without
comb, with or without stacking gel, etc.).
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