Sauer:bis-Tris SDS-PAGE, the very best: Difference between revisions

1X bis-Tris gel buffer, acrylamide solution to 5 %, water for remainder. This mixture can be stored alongside the resolving mix and APS as a "kit".

Optional: Add Bromophenol Blue to make the stacking gel blue.  It really helps when loading samples and doesn't affect the performance. You can fuss over the concentration, or just add it from a stock such that the gel in the casting has a decent blue color.  

I aliquot 2.5 mLs stacking per gel, which allows excess.

Don't add APS until you are ready to start the polymerization.  With this gel chemistry, the APS will start to polymerize the stacking gel if your resolving gel sets for a while and will be all goopy and unusable.

Add 15-20 μL 10% APS per gel, mix.

Add 10 μL TEMED per gel, mix, pour to fill the casting until slightly overflowing, insert the comb and leave the excess. Don't jam the comb all of the way in. The Bio-rad combs have small tabs that are intended as gap spacers.

Let polymerize. You'll notice a change in refraction as the wells polymerize up between the teeth of the comb. The polymerization typically stops before the surface and will not proceed to directly contact the comb (leaving rounded edges). Older protocols ask you to degas the solutions to promote polymerization, we found that this allows better polymerization, but accelerates the setting time to variable degrees, making it difficult to predict gel behavior. This is one reason to leave the excess solution on top.

Rinse with water to remove unpolymerized acrylamide.  Remove comb. Be careful here, go slow. Creating too much suction or pushing the comb back in will cause the well spacers to unseat and bend. If this happens, you can carefully adjust their position with a gel loading tip once the system is set up. However, samples can then leak between lanes, so don't use it for a Western. Once assembled, carefully rinse unpolymerized acrylamide from the wells with a needle and syringe. We cut the tip off of the needle with scissors and reuse the same one over and over.

 

If your system allows for it, rinse the casting with water before assembling the system (to remove unpolymerized acrylamide), and then remove the comb while the gel is in the tank covered with running buffer. This allows for the tank buffer the rush into the wells and rinse them and also lessens the chance of getting bubbles stuck in them.