Acrylamide gel preparation - for SDS-PAGE (May/17/2007 )

Hello, I have some basic questions about Acrylamide gel preparation:

1. I would like to use a disposable 10ml/50ml tube for preparing the gel solution, but I'm worried that the surface area will be too small for degassing. Must I use an Erlenmeyer flask?

2. I'm buying acrylamide solution, but the solution, TEMED, and APS still seem like dangerous stuff. Must I prepare and cast the gel in a fume hood? When cleaning the plates etc., should I be concerned about monomers and take any precautions beyond wearing gloves?

3. Can I dispose of the polymerized gel in standard trash, or is it hazardous waste?

Thanks for any advice.

---

When dealing with TEMED or DTT, yes, do it in fume hood. And of course when cleaning the plates, use gloves. Well, they claimed that polymerised gel will be nothing at all. So you can basically throw in a bin.

---

I usually do it in a 50 mL centrifugation tube. It's fine. you can also use the tube several times. try not to have foam on the surface of the liquid, unless you will aspirate a lot under vacuum. mix very gently. For most of purpose, I don't degas. I mix very very gently.


you can dispose the polymerized gel in standard trash, but only the polymerized gel. Sometime the gel left in the 50 mL tube doesn't polymerize so well, so I add some APS to the acrylamide left after pooling the gel, so it's perfeclty polymerized and I can throw it away.

---

Dear Ra!

Here is some suggestions arised from my experience

I prefer to degass gel solution ( resolving ) to enhance band resolution ( keep in mind that many other factors influence resolution like sample preparation, and this is only one but important ). Only degass before adding SDS to prevent intensive foam formation. don,t worry about 10-50ml tubes - I usually degass in these tubes. For fast degasation put magnetic stirrer into solution because viscosity varied depends on %acrylamide in working solution and decrease rate of degasation. I only not recommend you to degass working solutions when you prepare gradient gels. Because one of mixing solutions usually have high % acrylamide ( near 20%) and if you remove air bubbles you remove so main polymerisation inhibitor oxygen and rate of polymerisation will increase and may clogg your mix chamber ( several years ago I had this problem).

Concerning purchasing acrylamide solution. I prefer to prepare fresh stock ( every month) from solid acrylamide. Because duringacrylamide solution storage acrylic acid concentration increase. It is also decrease resolution of your PAAG. But you can solve this problem by incubating your acrylamide solution with Amberlite MB 150 resin ( Supelco or analog in Merck or Fluka). Store your stock with this resin ( covering bottom of storage bottle) in dark bottle at 4C.

Concerning dangerous of APS . Don't worry about this (APS) and work in lab without hood. About Temed - it is only amine with strong smell and so only don't leave it open after using ( aliquote it into eppendorf tubes under hood if you buy 1L bottle and then keep in 4C and work easy).

GOOD LUCK! and don't big worry about this!

---

Thanks for the suggestions everyone, this is helpful. I'll try my first SDS-PAGE gel today.

---