Stacking gel solution (3.75%) for SDS-PAGE (18.5 cm x 20 cm gels)
Prepare: the comb, the flask with rubber stopper and box with ice. Keep a flask on ice during the preparation of solution.
For the gels of 1 mm thickness:
|
|
20 ml (2 gels) |
10 ml (1 gel) |
|
Acrylamide/Bis-acrylamide (stock solution 30, w/v; crosslinker ratio 29:1; BIO-RAD, Cat.No: 161-0156) |
2.5 ml |
1.25 ml |
|
TRIS-HCl 2M, pH 6.8 (0.149 M final) |
1.49 ml |
745 microl |
|
Distilled H2O |
15.77 ml |
7.885 ml |
|
SDS 10 (0.12 final) |
240 microl |
120 microl |
Degas under vacuum for 15 min (do not forget to keep the flask with solution on ice). Then add:
|
TEMED (N,N,N,N’-Tetra-methylethylenediamine; BIO-RAD, Cat.No. 161-0800) |
72 microl |
36 microl |
|
Ammonium persulfate 10, w/v (0.06 final) |
120 microl |
60 microl |
Fill the gel cassette with stacking gel solution and insert a comb (avoid air bubbles), and allow for polymerization (at least 30 min). When a gel is polymerized, cover the top of the gel with Parafilm and put it into a cold room overnight for better polymerization.
A comb is used to generate wells for sample loading. Depending on the application, analytical (10-16 wells) or preparative scale (1-2 wells) combs can be used. The effective stacking gel height is 1.5 cm.
The gel may be stored for up to two weeks (at 4C, fully immersed in ‘running buffer’). Prolonged storage could result in dissolution of the sharp pH boundary between the lower pH (8.8) and upper pH (6.8) buffers.
Solutions: Tris T-1378 ‘Sigma’ 24.22g/100ml H2O, pH 6.8 with HCl
2002 Julius