Native Gel Electrophoresis

First, make up the stock solutions

1. Acrylamide solution: 30 grams acrylamide and 0.8 grams of bis-acrylamide. Fill up to 100 ml in distilled water and filter. The solution is stable for months at 4 degrees.

2. Separating gel buffer: 1.5 M Tris HCl, pH = 8.8

3. Stacking gel buffer: 0.5 M Tris HCl, pH = 6.8

4. Polymerizing solutions: 10% ammonium persulfate in water and TEMED

5. Sample buffer (5x):

15.5 ml of 1M Tris-HCl pH=6.8
2.5 ml of a 1% solution of bromophenol blue;
7.0 ml of water; and
25 ml of glycerol.
If you have a strong buffer in your sample please be sure that the final sample pH is 6.8.

6. Electrophoresis buffer: Dissolve 3.0 g of Tris base and the 14.4 grams of glycine in water and adjust the volume to 1 liter. The final pH should be 8.3.

7. Protein stain and destain: A standard Coomassie stain / destain will work to stain native gels.

Now pour and run the gel:

First pour the separating gel; To prepare the separating gel mix the following in a flask: 7.5 ml stock acrylamide solution, 7.5 ml separating gel buffer, 14.85 ml water, and 150 ul 10 percent ammonium persulfate. And 15 microliters of TEMED and gently swirl the flask to ensure even mixing. Pour the gel as you would a standard SDS-PAGE gel. Allow 30 minutes for the gel to polymerize. While the separating gel is polymerizing prepare the stacking gel solution: Mix the following in a flask: 1.5 ml stock acrylamide solution, 3.0 ml stacking gel buffer, 7.4 ml water, and 100 microliters 10 percent ammonium persulfate. Again, pour this gel as you would a standard SDS-PAGE. Now load your sample dissolved in the sample buffer described above. Attach the power leads so that the proteins are running towards the anode. Power up the gel at a current of 20 to 25 mA constant current. Run the gel until the bromophenol blue dye front reaches the bottom. (About three hours) Stain as you would a standard Coomassie gel. You can change the above concentration of acrylamide to separate out proteins for different molecular weight ranges. Also, be careful when running the gel as the gel may heat up and thus change your proteins biophysical characteristics.