PAGE polymerization problem - (Oct/25/2007 )
Recently, when pouring PAGE gels, a liquid layer develops on top of the gel, in both the separation and stacker gels. This layer is denser than the butanol I top my separating gel with. This is not detrimental to the separating gel, but a liquid layer at the top of the stacker forms horrible wells.
I have purchased new Acrylamide/bis mixture (30%/.8%), 10% APS, and TEMED. Still, this liquid layer appears, and polymerization takes 2 hours.
Has anyone seen this phenomenon before?
It is nature. O2 in the air supress the polymerization process, so you will have some leftover or less polymerized gels on top. In addition, gel contraction may also squeeze some water out. The way I solve the issue of soft top layer in the stacking gel is to simply add more gel solution such that un-polymerized, or poorly polymerized portion is way on the top.
It's a pain to have good wells in such conditions. You should increase the amount of APS or temed in your stacking gel.
I use 50 uL of APS10% and 3 uL of temed for 3 mL of stacking gel.
have you tried degassing? It might help to polymerise faster.
Tried degassing, and increased amounts of APS, TEMED, Bis-acrylamide in separate tests, tried adding APS an hour before TEMED to get a jumpstart on polymerization, and tried increasing the ambient temperature. Still, no luck.
adding extra bis-acrylamide will only increase crosslinking and make your stack more restrictive.
adding aps an hour before temed will do nothing but allow some decomposition of the aps. aps is the initiator, temed is the catalyst. without temed or a suitable replacement (eg-ampholytes) you will see no polymerization.
we use the formation of the liquid layer on the separating gel to tell us when the gel is polymerized. if you use a clean, dry comb with the stacking gel then the liquid layer will form at the top of the "teeth" created by the comb. the wells will be in proper shape when the comb is carefully removed. you must also allow enough time for (near) complete polymerization. we like to pour our stack well before we load the gel (an hour or two).
Thanks for the suggestions, all!
I increased the amount of TEMED, and upped the percent Bis- to 1.5, and raised the ambient temperature by about 4 degrees. Now I have no liquid phase at all, and my proteins separate beautifully even in denser crosslinker.
I thought adding too much TEMED or APS isnt a good idea right? Not too sure.
Adding too much TEMED will make the gel solidifies too fast. APS is an oxidant and only needed at catalytic amount.
better add isopropanol on the top of resolving gel. Butanol is more toxic (not the point ok..) but has a very low density to many compounds. So iPrOH is better and less hydrophobic. If aqueous layer still form you may also use ethanol 100%.