4% / 10% Stacking and Separating Protein Gel
Into a 15 mL centrifuge tube, add 5 mL 2x Separating Gel Buffer. 
Add 2.5 mL 40% Acrylamide/bis-acrylamide (29:1). 
Add 2.5 mL DI H2O 50 uL 10% Ammonium persulfate. 
Add 5 uL TEMED, then invert tube several times to properly mix all components and initiate the polymerization process.
Pipette 1 mL at a time of the 10% separating gel mixture between the two pieces of glass until you are about 75% of the way full.
Add a miniature amount of isopropyl alcohol on top of your separating gel so that it levels off (less than 50 uL), then wait for the gel to polymerize.
You will know it has polymerized as you should have some left in your centrifuge tube.
If you invert your centrifuge tube and the solution stays stuck on the bottom, then the gel has successfully polyermized.
To a new 15 mL centrifuge tube, add 2 mL of 2x stacking gel buffer.
Add 0.4 mL 40% acrylamide/bis-acrylamide (29:1). 
Add 1.6 mL H2O Add 20 uL 10% Ammonium Persulfate. 
Add 4 uL TEMED and invert tube to properly mix and initiate polymerization.
Use a paper towel to remove any leftover isopropanol, then fill the gel the rest of the way up by pipetting in 1 mL of the 4% stacking gel at a time until the top of the glass is reached, and place the comb in, ensuring that no air bubbles are created.
Let polymerize, then either use or store wrapped in a wet paper towel and plastic at 4 C.
