MiniProteanThreeRecipe

Recipes for the Bio-Rad Mini Protean III SDS-PAGE system

See also the long MiniProteanThreeInstructions without recipes.

The percentage of the resolving gel determines the molecular weights of proteins which can be resolved. 12% is a good generic gel to use and will resolve proteins from 15 kDa to 200 kDa with emphasis on resolving proteins from 15 kDa to 100 kDa. Lower percentage resolving gels will do a better job of resolving larger proteins, but will allow smaller molecular weight (MW) species to migrate off the gel. Higher percentage gels will similarly give extra resolution to lower MW species at a sacrifice of higher MW resolution.

resolving gel
6%
8%
10%
12%

40% acrylamide (29:1 crosslink)
600 μl
800 μl
1.0 ml
1.2 ml

4x resolving buffer pH 8.8
1 ml
1 ml
1 ml
1 ml

H₂O
2.4 ml
2.2 ml
2.0 ml
1.8 ml

10% APS
20 μl
20 μl
20 μl
20 μl

TEMED
2 μl
2 μl
2 μl
2 μl

Total volume
4.0 ml
4.0 ml
4.0 ml
4.0 ml

actually use 3.5 ml per gel
carefully layer water on top of the liquid gel after it has been poured and before the acrylamide has polymerized to ensure an evenly flat top to the stacking gel
polymerize for 45 minutes to 1 hour
- do not use less time even if the gel appears to have completed polymerization

stacking gel
one gel
two gels

40% acrylamide (29:1 crosslink)
117 μl
234 μl

4x stacking buffer pH 6.8
300 μl
600 μl

H₂O
783 μl
1566 μl

10% APS
12 μl
24 μl

TEMED
2 μl
4 μl

Total volume
1200 μl
2400 μl

actually use about 1 ml of mix per gel
be very careful to eliminate all air bubbles since oxygen inhibits acrylamide polymerization

The Mini Protean III needs about 350 ml of running buffer:

70 ml 5x SDS electrophoresis buffer
280 ml H₂O

be sure to rinse out the wells with running buffer after you assemble the gel into the running apparatus
boil samples in SDS loading buffer for two minutes and give a quick microcentrifuge spin-down immediately prior to loading
run the gel at 200 V constant voltage until the tracking dye has just finished migrating out of the bottom of the gel

resolving gel	6%	8%	10%	12%
40% acrylamide (29:1 crosslink)	600 μl	800 μl	1.0 ml	1.2 ml
4x resolving buffer pH 8.8	1 ml	1 ml	1 ml	1 ml
H₂O	2.4 ml	2.2 ml	2.0 ml	1.8 ml
10% APS	20 μl	20 μl	20 μl	20 μl
TEMED	2 μl	2 μl	2 μl	2 μl
Total volume	4.0 ml	4.0 ml	4.0 ml	4.0 ml

stacking gel	one gel	two gels
40% acrylamide (29:1 crosslink)	117 μl	234 μl
4x stacking buffer pH 6.8	300 μl	600 μl
H₂O	783 μl	1566 μl
10% APS	12 μl	24 μl
TEMED	2 μl	4 μl
Total volume	1200 μl	2400 μl