Why won't my SDS-PAGE stacking gel polymerize?
October 23, 2012 6:34 PM Subscribe
LongShotFilter: My SDS-PAGE stacking gel will refuses to polymerize. Copious amounts of fresh APS and TEMED are being used. I've spent a week on this and desperate measures are needed so the rest of my lab work can progress.
My lab manager has decided we will now be pouring our own SDS-PAGE gels. I pulled out the years-old equipment and tasked myself with figuring out how to do this. The resolving gel polymerizes nicely, but the stacking gel gets no farther than goo (and sometimes not even that far).
I'm using 12% resolving gel, 5% stacking gel. Recipes below:
Resolving gel (12%, 1ml solution) - does polymerize:
334ul DI H2O
250ul 1.5M Tris, pH 8.8
400ul 30% acrylamide/0.8% bis-acrylamide
5ul 20% SDS
10ul 10% APS
1ul TEMED
Stacking gel (5%, 1ml total) - Does not polymerize
549ul DI H2O
260ul 0.5M Tris, pH 6.8
170ul 30% acrylamide/0.8% bis-acrylamide
5ul 20% SDS
10ul 10% APS
1ul TEMED
Now, I tried doubling and tripling the amount of APS and TEMED (decreasing the water accordingly). I even set up a test series where I did 5x, 10x, and 20x APS and TEMED. Still no polymerization.
Solutions are at room temperature. APS is from a fresh bottle and made fresh with each attempt. TEMED is fresh. The only thing not fresh are the dry Tris, acrylamide, and bis-acrylamide reagents. The solutions themselves are fresh. Could the acrylamides have gone bad? But like I said, the 12% resolving gel is polymerizing.
Solutions are made in a yellow-light room, but when put out in white light polymerization is still unsuccessful.
Any experienced gel-makers have ideas?
My lab manager has decided we will now be pouring our own SDS-PAGE gels. I pulled out the years-old equipment and tasked myself with figuring out how to do this. The resolving gel polymerizes nicely, but the stacking gel gets no farther than goo (and sometimes not even that far).
I'm using 12% resolving gel, 5% stacking gel. Recipes below:
Resolving gel (12%, 1ml solution) - does polymerize:
334ul DI H2O
250ul 1.5M Tris, pH 8.8
400ul 30% acrylamide/0.8% bis-acrylamide
5ul 20% SDS
10ul 10% APS
1ul TEMED
Stacking gel (5%, 1ml total) - Does not polymerize
549ul DI H2O
260ul 0.5M Tris, pH 6.8
170ul 30% acrylamide/0.8% bis-acrylamide
5ul 20% SDS
10ul 10% APS
1ul TEMED
Now, I tried doubling and tripling the amount of APS and TEMED (decreasing the water accordingly). I even set up a test series where I did 5x, 10x, and 20x APS and TEMED. Still no polymerization.
Solutions are at room temperature. APS is from a fresh bottle and made fresh with each attempt. TEMED is fresh. The only thing not fresh are the dry Tris, acrylamide, and bis-acrylamide reagents. The solutions themselves are fresh. Could the acrylamides have gone bad? But like I said, the 12% resolving gel is polymerizing.
Solutions are made in a yellow-light room, but when put out in white light polymerization is still unsuccessful.
Any experienced gel-makers have ideas?
Ditto on quietgal - I would try layering with butanol over the top.
posted by pombe at 8:44 PM on October 23, 2012
posted by pombe at 8:44 PM on October 23, 2012
Also, if you haven't been pouring your own gels until recently, how old are the chemicals?
posted by Good Brain at 12:32 AM on October 24, 2012
posted by Good Brain at 12:32 AM on October 24, 2012
Ask the lab manager to compare the cost savings vs. the amount of money wasted paying you to try to get this up and running, and the work lost by the delays. The savings are probably already more than offset.
I still make a lot of my own solutions and etc., but when it comes to this kind of thing my lab group buys precast gels. No safety worries about unpolymerized acrylamide, no wasted time, better consistency and reproducibility.
posted by caution live frogs at 6:52 AM on October 24, 2012 [1 favorite]
I still make a lot of my own solutions and etc., but when it comes to this kind of thing my lab group buys precast gels. No safety worries about unpolymerized acrylamide, no wasted time, better consistency and reproducibility.
posted by caution live frogs at 6:52 AM on October 24, 2012 [1 favorite]
Response by poster: Purchasing is not an option, manager would prefer us spend the time getting it running to buying the gels.
I layered not-water saturated butanol over it but it didn't make a difference. Should I try water-saturating it first?
posted by Anonymous at 7:44 AM on October 24, 2012
I layered not-water saturated butanol over it but it didn't make a difference. Should I try water-saturating it first?
posted by Anonymous at 7:44 AM on October 24, 2012
A mix of comments and questions that may or may not be helpful:
-Are you mixing the gel solutions? I do invert my Falcon tubes to get the APS and TEMED mixed in a bit, but you don't want to aerate the gel mix with, say, vortexing.
-You say "APS bottle" - I assume you mean a container with dry APS, right, not a solution? (Because it totally does go bad when aqueous, and I make it fresh from powder every time because life's too short to spend re-pouring stupid gels.)
-How long are you waiting for the stacking gel to polymerize? A little longer doesn't necessarily hurt (heck, overnight polymerization is even preferable in some ways, though it can be inconvenient and isn't necessary for standard SDS-PAGE setups.) And a stacking gel will always be more gooey than a resolving gel - you can observe this when you take things apart after a run so that you can stain the gel.
-You say "solutions are at room temperature." Have you been storing your acrylamide solution at room temperature? The solution should be stored at 2-8°C. (In the dark, preferably, unless you go through it fast.)
-Have your acrylamide powders been stored in the dark (for plain acrylamide)? At 2-8°C (for the N,N′-Methylenebis(acrylamide))?
-Your volumes seem pretty small, and with a high stacking to resolving gel ratio. If you're using something other than more-or-less standardly sized mini gels (e.g. the ones you'd make for a Bio-Rad Mini-Protean rig), might that be relevant?
While yeah, you can degas your buffers, I have never needed to do so except for DNA sequencing gels, and I have never needed to layer the tops of my stacking gels with butanol or isopropanol. You shouldn't need to resort to extreme measures for standard protein SDS-PAGE. If I had to bet, I would bet on your acrylamide being iffy (either due to storage conditions/age of the powders or the solution), and that the problem is worse in the stacking gel because there is less acrylamide in it. Can you mooch a few mLs of premixed acrylamide solution off of a nearby lab that does gels regularly, or buy 100mL from Sigma or someone (since it looks like you are pouring pretty standard gels, not high C% Schägger gels or whatever)? That would at least confirm or refute the possibility that your acrylamide is at fault. Ditto for the Tris, if you're feeling paranoid.
posted by ubersturm at 9:36 AM on October 24, 2012 [1 favorite]
-Are you mixing the gel solutions? I do invert my Falcon tubes to get the APS and TEMED mixed in a bit, but you don't want to aerate the gel mix with, say, vortexing.
-You say "APS bottle" - I assume you mean a container with dry APS, right, not a solution? (Because it totally does go bad when aqueous, and I make it fresh from powder every time because life's too short to spend re-pouring stupid gels.)
-How long are you waiting for the stacking gel to polymerize? A little longer doesn't necessarily hurt (heck, overnight polymerization is even preferable in some ways, though it can be inconvenient and isn't necessary for standard SDS-PAGE setups.) And a stacking gel will always be more gooey than a resolving gel - you can observe this when you take things apart after a run so that you can stain the gel.
-You say "solutions are at room temperature." Have you been storing your acrylamide solution at room temperature? The solution should be stored at 2-8°C. (In the dark, preferably, unless you go through it fast.)
-Have your acrylamide powders been stored in the dark (for plain acrylamide)? At 2-8°C (for the N,N′-Methylenebis(acrylamide))?
-Your volumes seem pretty small, and with a high stacking to resolving gel ratio. If you're using something other than more-or-less standardly sized mini gels (e.g. the ones you'd make for a Bio-Rad Mini-Protean rig), might that be relevant?
While yeah, you can degas your buffers, I have never needed to do so except for DNA sequencing gels, and I have never needed to layer the tops of my stacking gels with butanol or isopropanol. You shouldn't need to resort to extreme measures for standard protein SDS-PAGE. If I had to bet, I would bet on your acrylamide being iffy (either due to storage conditions/age of the powders or the solution), and that the problem is worse in the stacking gel because there is less acrylamide in it. Can you mooch a few mLs of premixed acrylamide solution off of a nearby lab that does gels regularly, or buy 100mL from Sigma or someone (since it looks like you are pouring pretty standard gels, not high C% Schägger gels or whatever)? That would at least confirm or refute the possibility that your acrylamide is at fault. Ditto for the Tris, if you're feeling paranoid.
posted by ubersturm at 9:36 AM on October 24, 2012 [1 favorite]
Best answer: I don't actually make only 1ml gels, those are just the recipes for 1ml to make it easier to compare the components. The stacking gel was left to polymerize overnight, and it was too gooey for any wells to form, period.
think I solved this one. The acrylamide powders were not stored in the dark and are about seven years old. I borrowed some fresh pre-made acrylamide/bis-acrylamide solution and everything polymerized just fine. All the info I looked at online addressed old APS and TEMED, but it looks like acrylamide can go bad too. Thanks all!
posted by Anonymous at 2:15 PM on October 24, 2012
think I solved this one. The acrylamide powders were not stored in the dark and are about seven years old. I borrowed some fresh pre-made acrylamide/bis-acrylamide solution and everything polymerized just fine. All the info I looked at online addressed old APS and TEMED, but it looks like acrylamide can go bad too. Thanks all!
posted by Anonymous at 2:15 PM on October 24, 2012
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How are you keeping air away from your stacker? Does the comb completely cover the top of the acrylamide solution? If any of it is exposed to air, you can carefully layer water-saturated butanol over the top with a pasteur pipet; it will float and provide a barrier while the acrylamide polymerizes, then you can pour it off and rinse the gel with a little running buffer before loading your samples.
posted by Quietgal at 7:45 PM on October 23, 2012 [1 favorite]