5
$\begingroup$

I am working on 20% SDS PAGE. I want to know optimum polymerization time for 20% resolving gel and 6% stacking gel. If I increase the time then would it affect the band pattern?

$\endgroup$
  • 1
    $\begingroup$ it depends on many factors such as APS concentration, TEMED concentration and ambient temperature $\endgroup$ – WYSIWYG Dec 23 '14 at 7:35
3
$\begingroup$

APS and TEMED concentrations matter a lot. Even with that standardized, your polyacrylamide on the shelf is losing reactive groups as we speak. Who know how long it sat in the warehouse? It all adds up to the fact that the minimum required time is practically impossible to predict.

On the other hand, once polymerized, a gel is stable for days without apparent loss of quality. Overnight polymerization will be "optimal" in the sense that the gel won't get any better with longer or shorter polymerization. But so will be a two-days old gel. People used to cast gels every Friday, and leave them in wet paper towels in the cold room for the next week.

$\endgroup$
9
$\begingroup$

There is nothing like a general polymerization time, as this is a chemical reaction which depends on many factors. The most important are:

APS: Concentration and and the age of the chemical are important. APS decomposes over time when it is not stored completely dry (which it is not on most benches) and also with freezing thawing cycles in storage. APS provides the free radicals necessary for the reaction; without it no polymerization will happen. In my experience APS is the most critical part of the reaction, with old solutions making the polymerization really slow. I would make a stock solution, freeze it in aliquots. For making gels I thaw on aliquot and keep it in the fridge afterwards for a week and then discard it.

TEMED: Is the catalyst for the reaction, too much of it will start the reaction very fast.

Temperature: The warmer it is, the faster the polymerization will be. In the cold room you will not get complete reaction, if any.

What you can do is to store the remaining acrylamide in the beaker and wait until it is polymerized. Then your gel should also be ok to use. A longer polymerization time (assuming that your gel was completely polymerized before) will not change the resolving capacity of your gel.

$\endgroup$
  • $\begingroup$ "with old solutions making the polymerization really fast"... I guess you meant fresh solutions. Old solutions are bad. $\endgroup$ – WYSIWYG Dec 23 '14 at 10:52
  • $\begingroup$ -1 For having to scrape acrylamide out of the beaker! (I didn't actually down vote) $\endgroup$ – canadianer Dec 24 '14 at 0:17
  • $\begingroup$ @canadianer You can do the mixing in a plastic tube if you want and discard it when the polymerization is finished. Makes more trash though. $\endgroup$ – Chris Dec 24 '14 at 8:18
6
$\begingroup$

Others have already explained the effect of different factors on polymerization time. This answer is mostly about:

If I increase the time then would it affect the band pattern?

It may.

Some tips:

  • Make a little extra mix so that you can leave some in the beaker to know if polymerization has happened.
  • Always layer the poured resolving gel with water so that the gel doesn't get dried up.
  • Don't leave the gel for long time after polymerization. Not only it wastes time, it also may cause the gel to dry up and SDS/Urea to precipitate/crystallize.
  • Wash the gels with the running buffer after removing the comb/before loading the sample. You can use a fine needled (high gauge) to do that.
  • Pre-run the gel to equilibriate it; this not generally necessary and as Nick Sandor said, would destroy stacking gel. However it is important for big urea gels and somewhat important for continuous SDS-gels if you have left it for a quite some time post polymerization. So it is better to not leave the gel for a long time
$\endgroup$
  • 2
    $\begingroup$ In theory, pre-running SDS-PAGE discontinuous gels will destroy the stacking gel, because glycine, which doesn't belong there, will go in. In practice, I see a lot of people recommending it, regardless of theory, and in the absence of any empirical evidence. "To remove unpolymerized acrylamide", some say, to which I reply that it will take just as much as passing bromophenol blue and glycine through the gel. $\endgroup$ – Nick Alexander Dec 23 '14 at 23:11
  • $\begingroup$ @NickSandor For usual SDS-PAGE it is not necessary- I agree and thanks for the point; it would change the pH and composition of stacking gel. I edited the answer. But for urea gels (continuous) it is always recommended. $\endgroup$ – WYSIWYG Dec 24 '14 at 4:41
4
$\begingroup$

The polymerization time is strongly dependent on APS and TEMED concentrations. I do not think that the increased time for polymerization would somehow influence the division of the proteins in negative way. Actually the time varies from 20 min to 1 h, but I personally leave the gel to polymerize longer. Unpolymerized Acrylamide could build adducts with some proteins which could result in multiple bands. In addition acrymamide itself is toxic, so I always give it additional time to polymerize. Good luck!

$\endgroup$

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.