16 Preparing buffers for polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis (PAGE) separates molecules in complex mixtures. Gel pore size is a function of the acrylamide concentration used, and higher concentrations of acrylamide are used to resolve smaller molecules. You can use either a single-percentage gel, or you can have gradient gels that have a range of acrylamide concentrations within a single gel (such as ~4-20% acrylamide). To separate proteins ONLY by size and not by charge, you will place an ionic detergent in both the gel running buffer and the sample buffer. The detergent commonly used is sodium dodecyl sulfate (SDS). Most proteins bind SDS in such a way that they migrate in a gel as if they have nearly identical charge (all negative), so mass becomes the only factor in determining the migration rate of the protein.

You’ll first prepare the buffers you need to run your SDS-PAGE gels. These protein buffers have a different composition than the solutions we used to separate DNA.

You will run your protein gels using: 1X SDS-PAGE Tris-Glycine Running Buffer (TGS buffer)

The final composition of 1X TGS buffer is:

• 25 mM Tris base
• 192 mM glycine
• 0.1% SDS

To make our 1X buffer, we will first prepare 10X Tris-Glycine buffer and a solution of 10% SDS. These will be diluted in di H20 to obtain our 1X TGS buffer. You’ll use more of the 10X Tris-Glycine buffer when you perform your Western blot.

Each group of 4 students will make of 300 mL of 10X Tris-Glycine, which will contain:

0.25 M Tris base

1.92 M glycine

 First, perform the calculations to determine how much of each reagent is necessary to make 300 mL of this 10X Tris-Glycine solution. Check your math with your instructor before making your buffer.

Values you’ll need:

Tris molar mass = 121.14 g/mol

Glycine molar mass= 75.07 g/mol

Then, prepare the supplies you’ll need

• deionized water
• Tris Base, electrophoretic grade
• Glycine, electrophoretic grade
• graduated cylinder to fit the final volume you are making
• pyrex beaker (at least 2X your final volume)
• polypropylene or glass bottle to store your finished solution
• Magnetic stir bar
• Magnetic stir plate

Protocol for TGS buffer preparation.

1. Measure out ~75% of the water volume you’ll use (use the graduated cylinder) and pour into the pyrex beaker.
2. Add stirbar, place on stirplate, and begin stirring.
3. GRADUALLY add Tris-Base and then glycine. Add these reagents one by one, and do so gradually. VERY SLOWLY adding the powders to water that is being stirred makes them dissolve in water much faster.
4. Stir ~20 minutes to ensure that all reagents are completely dissolved.
5. Dilute from 10X to 1X before use.

Making 1X TGS from 10X Tris-Glycine

To run a gel, each group (of four) needs to make up 750 mL of 1X TGS (Tris-glycine with added SDS). Combine your 10X Tris-Glycine buffer with 10% SDS and diH20 to obtain the final concentrations below.