Hi there,

An easy way to check the polymerization of the gel is to save a bit of the solution you have just poured and follow its polymerization. If it has polymerized in the tube (in the presence of air which is inhibiting the process), it has  also polymeryzed between the gel plates.

One hour is definitely a long time for polymerization...

Hi M. Manirujjaman

It should be not take that long time for separating gel to have complete polymerization, normally it only takes around 20-30 min. But it is also depends on how thick your gel is. One hour for polymerization is more than enough, and you can use it for your experiment.

Here attached the procedure from UCLA, that I usually used.

Best luck

-Fean-

If you think it takes too long, you can always adjust your concentrations of TMED and APS to get quicker polymerisation. 

Hello Manirujjaman,

TEMED and APS (10%) with help to polymerise the gels quickly said by Dr. Thibaut. But once you cast the gels (Both resolving and stacking), wait for atleast 1 hour before loading the samples into the gels. This will provide sufficient time for both the gels to polymerise. Furthermore, keep the casted gels in running buffer for atleast 10-15 minutes before loading the samples.(This step will adjust the temperature difference between gel and running buffer).

Hi there,

An easy way to check the polymerization of the gel is to save a bit of the solution you have just poured and follow its polymerization. If it has polymerized in the tube (in the presence of air which is inhibiting the process), it has  also polymeryzed between the gel plates.

One hour is definitely a long time for polymerization...

I fully agree with Dominique and it is a common understanding that if its getting polymerized in the tube (in the presence of air), it will certainly polymerize in the casting plates(since there is no air).

If you have carried out all the all the steps properly it would not take more than 15-20 minutes.

Hi Manirujjaman,

If you are following protocol correctly, even then if it is taking long time for polymerization, that means either your TEMED is quite old or your not adding sufficient to the solution.

APS should be prepared freshly. Moreover you can check the concentrations as mentioned in standard protocol.

In my case, I do believe that APS and TEMED has a great impact of polymerization of SDS-PAGE gel. Sometime in the winter when the temperature is below 250 C, it takes long time.

What I do is already suggested by Dominique. I save at least 1 mL of the resolving and stacking gels in the tubes and monitor their solidification to assess the time of polymerization of the gels. The time taken for polymerizing depends on the concentration of the gel. As suggested you can always increase APS and TEMED to achieve faster polymerization. Usually, in my case, 6 mL resolving and 3 mL stacking gel takes not more than 20-30 mins to solidify. Check your acrylamide solution and use freshly prepared reagents.

Hope it helps!

In my experience, just a few minutes are more than enough for SDS-PAGE gel polymerization. Of course, it also depends on acrylamide % and APS and TEMED ammounts, but if it takes 1 hour.........you have a problem.

If you use Acrylamide which is made long time ago, it takes much time to form the gel

It depends on acrylamide % and APS and TEMED ammounts , when you  adjust the concentrations gel polymerization takes  a few minutes  . 

Prepare a fresh APS to get the better polymerization.

Add good quality TEMED to accelerate the polymerization.

It all depends on your Acrylamide, 10 % APS and TEMED quantities. I for separating gel I have used 2.5 ml of 30% Acrylaminde, 45 ul of 10% APS and 11 ul of TEMED. It takes 15 minutes for the gel to polymerise . For stacking gel i have used 355 ul of 30% Acrylaminde, 18 ul of 10% APS and 3.2 ul of TEMED. It also takes 15 minutes for the gel to polymerise.

If TEMED (light-sensitive) has a yellow-brownish appearance that means it'd been oxidised and you need to refresh it. Must save 10% APS solution in the fridge (2-4°C) and can last up to one year, usually 50 ml for public use is sufficient.

From my experience, if they are newly prepared, this step can be shortened within 10 min. You can touch the glass assemblies to feel the warmth to ensure they are working. It's a slow and exothermic reaction.

Tip: I only use dH2O at RT, since others remain cool, the mixtures won't be too warm nor cold lest you wait too long.

30 mins for me at RT for both resolving and stacking is a good indicator - may also depend on the thickness of your gel according to the plates and their leakiness if any

Good luck!

This is all good advice.  Another point.  The gel will polymerize more slowly if it is cold.  

Several years ago our gels were not polymerizing.  We found out someone had used a pipet tip that had been in APS to also pull out the TEMED.  The TEMED no longer worked very well.  Switching to another bottle of TEMED solved the problem.

What is the approximate voltage and time to run a (20% separating and 5%stacking ) SDS PAGE ?

It depend on the plate size.If I used big plate (Approximately 13X12 cm gel) and 10-12% gel initially I set up 300 V and 20 mA current.In the case of new buffer it takes about 3-4 hours to complete the separation.