1.3.2. Preparing and running vertical slab gels
This protocol is designed for a vertical slab gel with dimensions 100 x 120 x 0.9 mm (Fig. 2). For thicker gels or other gel sizes, the volumes of separating and stacking gels and the electric current must be adjusted accordingly.
1. Prepare 100 ml of an acrylamide stock solution containing
1.1. 30 % (w/v)
1.2. 0.8 % (w/v) N,N´-methylene bisacrylamide in ddH2O.
This solution can be stored refrigerated for 2-3 weeks.
2. Prepare a 1.5 M Tris buffer with a pH of 8.8, for this add:
2.1. 18.15 g of Tris-base to
2.2. 80 ml of ddH20.
2.3 Use 1N HCl to adjust pH to 8.8.
2.4. Complete volume to 100 ml.
3. Prepare a 0.25 M Tris buffer with a pH of 6.8, for this add:
3.1. 3 g of Tris-base to
3.2. 80 ml of ddH20
3.3 Use 1N HCl to adjust pH to 8.8
3.4. Complete volume to 100 ml.
These buffers for the preparation of the separation and stacking gels, respectively, can be stored refrigerated.
4. Prepare 1 l of electophoresis buffer: dilute in 1 l of distilled water
4.1. 3.03 g Tris.
4.2. 14.4 g glycine.
4.3. 1 g SDS.
A polyacrylamide gel with a 7.5 % separating gel is then prepared and run in the following sequence:
ml of the acrylamide/bis-acrylamide stock solution,
5.2. 5 ml of the 1.5 M Tris buffer (pH 8.8),
5.3. 2.3 ml ddH2O.
6. Stir gently so as not to introduce air bubbles, as oxygenation may impede polymerization.
7. Add 190 µl of 1 % ammonium persulphate (APS) solution and 40 µl of N,N,N′,N′-Tetramethylethylenediamine (TEMED).
These are the starters for the polymerization process.
8. Quickly mix the reagents and immediately pour the solution into the cassette formed by the two glass plates sandwiched over sealing spacers.
Leave sufficient space for later pouring the stacking gel on top.
9. Carefully overlay the gel with water to guarantee a smooth and straight surface.
10. Wait for about 30 min until the gel is completely polymerized.
If it does not polymerize in due time, your APS solution is probably too old.
11. After polymerization is completed, pour off the water and carefully remove any remaining water with filter paper, but avoid touching the gel surface.
12. Prepare a stacking gel (4.26 %) by mixing
12.1. 0.375 ml of the acrylamide/bisacrylamide
12.2. 1.38 ml of the 0.25 M Tris buffer (pH 6.8) .
12.3. 0.825 ml ddH2O.
13. Stir gently so as not to introduce air bubbles.
14. Add 51 µl of a 5 % ammonium persulphate (APS) solution.
15. Add 10 µl TEMED.
16. Pour the stacking gel on top of the separating gel.
17. Insert a Teflon comb with blunt-ended teeth for creating the sample application wells.
Be careful to avoid introducing air bubbles.
18. Allow the gel to polymerize completely at room temperature.
Once polymerized, gels can be stored in the refrigerator for up to a few days, but make sure to wrap the glass plate-gel sandwich with household PVC film.
19. Mount the gel sandwich in your vertical electrophoresis apparatus.
20. Fill the tanks with electrophoresis buffer.
21. Carefully remove the Teflon comb pulling up evenly by a small amount on each side.
22. Using a micropipette with long tips or a Hamilton-type syringe load the samples into the wells.
The sample solution will be more dense than the running buffer, and will displace the latter when pipetted into the comb wells; the added bromophenol blue allows this to be visualized.
23. Connect the electrophoresis apparatus to a power supply, make sure that polarity is correct (see Fig. 2).
24. Carry out the electrophoresis run at a constant current of 15 mA, preferably in a cold room or refrigerator, until the bromophenol blue front reaches the desired position, usually 0.5 cm above the gel bottom.
25. Switch off power supply and remove cables.
26. Dismount the cassette, remove the stacking gel and carefully slide the gel off the glass plate into the dish containing the fixation/staining solution.
General advice: Ammonium persulphate (APS) decomposes within a short time, so a fresh working solution should be prepared weekly. Both APS and TEMED are starters for the polymerization process, so make sure to pour gels quickly after these compounds are added.
User safety: Acrylamide and bisacrylamide are neurotoxic compounds, so use protective equipment (fume hood, safety glasses and gloves) when preparing and handling any acrylamide solution. Even though after polymerization, polyacrylamide is no longer toxic, some unpolymerized acrylamide residue is still present, so gels should always be handled with gloves.