Protocols

Tricine-SDS-PAGE

Posted on Friday, October 17, 2003

Description
Tricine-SDS-PAGE

Procedure
Assemble each gel sandwich by stacking, in order, the notched aluminum plate, two 0.75-mm spacers, and a glass plate. It is important that the spacers are aligned properly.

Fit the gel sandwiches (usually four at a time) tightly in the multiple gel caster. Fill any remaining space in the mold by including additional glass plates.

Place the front face plate on the caster, clamp it in place against the silicone gasket, and verify the alignment of the glass plates and spacers.

Prepare the separating gel solution as directed below. Do not add TEMED or ammonium persulfate until ready to pour.

Fill a 50-ml syringe with the solution and slowly inject it into the caster until the gels are 6 cm high, allowing 1.5 cm for the stacking gel.

Overlay each gel with 100 ul 0.1% SDS. Allow gels to polymerize for approximately one hour to overnight.

Remove any remaining gel overlay solution by blotting the top of each gel with a piece of Whatman 3mm paper.

Prepare the stacking gel solution as described below. Fill a 10-ml syringe with the solution, and inject into each gel sandwich until the top is reached.

Carefully place an appropriate comb into each gel, taking care not to trap any bubbles. Allow gel to polymerize for about 1 hour.

Remove the front faceplate of the caster. Carefully remove the gels, and separate using a razor blade.

The gels can be stored with the combs in place tightly wrapped in plastic wrap inside a sealable bag at 4°C for 2 to 3 weeks. Keep the gels moist. Do not store gels in the multiple caster.

When ready to run the gel, remove the comb and rinse the sample wells with cathode buffer (see below). Place a line indicating the bottom of the each well on the front glass plate, or use a well template.

Fill the upper (cathode) and lower (anode) buffer chambers with the appropriate buffer. The upper chamber should be filled to 1 to 2 cm above the notched plate.

Using the marks on the glass plate or the well template as a guide, pipette the prepared samples into the wells.

Electrophorese the samples at 10 mA constant current per 0.75-mm gel until the dye front reaches the top of the separating gel (45 minutes). Increase current to 20 mA per gel, and continue run until the bottom of the gel is reached (1 hour).

Recipes
Separating gel monomer (49.5% T 6% C)

Mix 93.0 g acrylamide and 6.0 g N, N'-methylene-bisacrylamide in a total volume of 200 ml dH2O. Filter solution through a 0.45 µm filter and store at 4°C protected from light. Discard after 30 days.


Stacking gel monomer (49.5% T 3% C)

Mix 96.0 g acrylamide and 3.0 g N, N'-methylene-bisacrylamide in a total volume of 200 ml dH2O. Filter solution through a 0.45 µm filter and store at 4°C protected from light. Discard after 30 days.


CAUTION: Acrylamide and bisacrylamide are potent neurotoxins and are absorbed through the skin. Wear a mask while weighing the powder. Gloves and a lab coat should be worn when handling the solution. Do not mouth pipette.


Anode buffer (0.2 M Tris, pH 8.9)

Dissolve 12.11 g Tris base in 200 ml dH2O. Adjust pH to 8.9 with 1 N HCl. Add dH2O to 500 ml total volume. Filter the solution through a 0.45 µm filter and store at 4°C.


Cathode buffer (0.1 M Tris, 0.1 M tricine, 0.1% SDS, pH 8.25)

Dissolve 6.055 g Tris base and 8.96 g tricine in a total volume of 500 ml dH2O. Filter the solution through a 0.45 µm filter, add 0.5 g electrophoresis-grade SDS, and store at 4°C. It is not necessary to adjust the pH of this buffer, which should be around 8.25.


Gel buffer (3.0 M Tris, 0.3% SDS, pH 8.45)

Dissolve 181.65 g Tris base and 1.5 g electrophoresis-grade SDS in 300 ml dH2O. Adjust the pH of the solution to 8.45 with concentrated HCl. Bring to final volume of 500 ml with dH2O. Store at 4°C.


Stacking gel (4.0% T 3.0% C)

In a 50-ml conical tube, mix 0.8 ml of the stacking gel monomer, 2.5 ml of the gel buffer, and 6.7 ml dH2O. Degas under vacuum 10 to 15 minutes. Add 50 µl of 10% ammonium persulfate and 10 µl TEMED. Swirl gently to mix. Use immediately. Produces 10 ml of stacking gel, sufficient for four minigels.


Separating gel (16.5% T 6.0% C)

In a 50-ml conical tube, mix 10.0 ml of the separating gel monomer, 10.0 ml of the gel buffer, 3.1 ml glycerol, and 6.9 ml dH2O. Degas under vacuum 10 to 15 minutes. Add 100 µl of 10% ammonium persulfate and 20 µl TEMED. Swirl gently to mix. Use immediately. Produces 30 ml of separating gel, sufficient for four minigels.



Supplies


Tips
Protocol adapted from Schägger, H., and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166:368-379, as modified by Lesse, A. J., et al. 1990. Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Immunol. Methods 126:109-117.

Submitted by: manny440