Bio-Rad PROTEAN® Plus Hinged Spacer Plates and Combs - Manual (Page 14)

b. The total volume required for multiple gels can be determined by multiplying the vol-
ume of a single gel by the number of gels required.
8.3 Gel Preparation
Monomer concentrations as high as 22% T can be prepared from a 30% T stock solution
as presented in Table 8.3. When monomer concentrations greater than 22% T are required, the
Acrylamide/Bis stock solutions must be increased to 40% T 0r 50% T.
Table 8.3. Formulations for SDS-PAGE Separating
and Stacking Gels
Separating Gel Stacking Gel
(0.375 M Tris, pH 8.8) (125 M Tris, pH 6.8)
Monomer Concentration (%T, 2.67%C) C 10% 20% X%(<15%) X%>(15%) 4.0%
Acrylamide/Bis (30%T, 2.67%C) Stock 33.3 ml 66.6 ml d ml d ml 1.3 ml
Distilled water 40.2 ml 6.9 ml e ml e ml 6.1 ml
1.5 M Tris-HCl, pH 8.8 25.0 ml 25.0 ml 25.0 ml 25.0 ml
.5 M Tris-HCl, pH 6.8 2.5 ml
10% (w/v) SDS 1.0 ml 1.0 ml 1.0 ml 1.0 ml 100 ml
10% ammonium persulfate (fresh) f 500 µl 500 µl 500 µl 500 µl 50 µl
TEMED f (monomer concentration <15%) 50 µl 50 µl 10 µl f
TEMED f,g (monomer concentration >15%) 25 µl 25 µl
TOTAL MONOMER h 100 ml 100 ml 100 ml 100 ml 10 ml
c. The pore size of the polyacrylamide gel can be changed by adjusting either the total
monomer concentration (%T) or by adjusting the crosslinking monomer concentration
(%C). The most common method of changing the pore size is to adjust the total monomer
concentration (%T). In diluting a stock solution, the crosslinking monomer concentra-
tion (%C) is independent of the final total monomer concentration (%T).
%T=[g Acrylamide + g Bis-Acrylamide/Total Volume] x 100
%C=[g Bis-Acrylamide/(g Acrylamide + g Bis-Acrylamide)] x 100
d. Calculate the volume of Acrylamide/Bis stock required for the desired total monomer
concentration with the following formula: volume 30% T, 2.67% C Stock = (X % T) x
(3.33). For example, the volume of Acylamide/Bis stock required to prepare 100 ml final
volume of 10% T monomer solution would be 33.0 ml; 10 x 3.33 = 33.3 ml.
e. Calculate the volume of water required for the desired total monomer concentration with
the following formula: volume water = 73.5 - (volume 30% T, 2.67% C stock used). For
example, the volume of water required to prepare 100 ml final volume of a 10% T
monomer solution would be 40.4 ml: 73.5 - 33.3 ml = 40.2 ml.
f. Prepare the monomer solution by combining all reagents except ammonium persulfate and
TEMED. Dearate the solution under vacuum for at least 15 minutes. Add the two cata-
lysts just prior to casting the gels.
g. Higher concentrations of monomer polymerize more evenly with lower concentrations of
TEMED.
h. One can prepare any desired volume of monomer solution by multiplying to 100 ml recipe
by the desired multiplying factor.
i. Higher TEMED concentrations and faster polymerization are required for the stacking
gel because of the inhibitory effect of atmospheric oxygen associated with the comb.
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