SDS PAGE with 40% Acryl and 2% Bis-Acrylamide
Materials:
- This volume enable you to pour 2 x ‘Bio-Rad’ SDS-Poly acrylamide gels + a little extra
Resolving gel:
Ingredient
10%
12.5%
15%
40% Acrylamide
2% Bis-acrylamide
1.5M Tris pH 8.8
10% SDS
MilliQ H2O
10% APS
TEMED
2.78 ml
0.36 ml
2.81
0.1 ml
5.05 ml
37 μl
24 μl
3.475 ml
0.45 ml
2.81
0.1 ml
4.267 ml
37 μl
24 μl
4.22 ml
0.49 ml
2.81 ml
0.3 ml
3.48 ml
37 μl
24 μl
Total
11.149 ml
11.149 ml
11.149ml
4% Stacking gel:
Ingredient
4%
40% Acrylamide
2% Bis-acrylamide
0.5M Tris pH 6.8
10% SDS
MilliQ H2O
10% APS
TEMED
0.77 ml
0.39 ml
0.75 ml
0.05 ml
3.2 ml
30 μl
9 μl
Total
5.199 ml
Protocol:
1. Mix components and pour resolving gel. You want to leave at least 0.35 mm under
the comb length.
(i.e. the well depth) so there is room for the stacking gel to effectively ‘stack’ your
sample, if not you may get poor resolution.
2. Overlay with MilliQ H2O and leave to set.
3. Pour off MilliQ H2O layer (not critical to remove every drop)
4. Pour stacking gel and place comb in between plates – avoid bubbles under the
wells.
Important notes
- Do NOT add 10% APS OR TEMED until you are ready to pour (this means
you may mix everything for the stack, but you don’t pour this part until resolving gel
has set).
- This protocol tends to result in very quick setting gels. Bear this in mind if you
up-scale.
- The ratio of 40% Acrylamide: 2% Bis-acrylamide is 8.61:1.