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 .
