Some suggestions that might help you with your problem:

- use freshly prepared APS. It can be re-used when stored at -20 degrees C but not too often.

- clean glas equipment that you use for preparing the gel carefully. Even tiny amounts of dust can prevent acrylamide polymerisation.

- do you store the Tris buffer and/or the acrylamide solution in a fridge? Before preparing the gel let the solutions warm to room temperature. If the solutions are too cold polymerisation takes too long.

- the acryalamide solution you use also contains bisacrylamide solution as a crosslinker? Beware that there are also acrylamide solutions that do not contain bisacrylamide.

- prepare your gels at room temperature.

- lastly: maybe your acrylamide solution is too old. Use a fresh one if possible.

Good luck!

Cheers, Christian

Some suggestions that might help you with your problem:

- use freshly prepared APS. It can be re-used when stored at -20 degrees C but not too often.

- clean glas equipment that you use for preparing the gel carefully. Even tiny amounts of dust can prevent acrylamide polymerisation.

- do you store the Tris buffer and/or the acrylamide solution in a fridge? Before preparing the gel let the solutions warm to room temperature. If the solutions are too cold polymerisation takes too long.

- the acryalamide solution you use also contains bisacrylamide solution as a crosslinker? Beware that there are also acrylamide solutions that do not contain bisacrylamide.

- prepare your gels at room temperature.

- lastly: maybe your acrylamide solution is too old. Use a fresh one if possible.

Good luck!

Cheers, Christian

Thank you very much Dear Christian. I will try to follow your suggestions in the next time.

hi,

i agree with Christian that the freshness of the APS is vital ( some make up fresh APS for every gel or at least every week ) as is excluding air by covering the gel while it polymerises..oxygen inhibits the polymerisation which is why some people degas their reagents but this is not necessary but coover the part of the gel exposed to the air with clingfilm while setting.. I would use 40 ul APS and 4 ul of temed ( very stable temed never goes off) for this composition of gel. You can test the gelling time by putting some in a sealed eppendorf tube to assess how long you have to pour the gel before it sets. Look just under the open end of the gel....sometimes a little liquid is squeezed out of the gel which has set but just on top there is liquid which looks like it has not set. you should see the interface where gelling has taken place

Thank you very much Dear Paul

Me too agreed with Dr. Scheckhuber . Do use freshly prepared APS.

Crystals of APS tend to collect water and become useless. When you add water to APS you should hear hissing sound while APS is diluting and also some very small bubbles should appear. If there are no bubbles, than crystals might not be useful anymore. However, all this replied to you so far could be in vain, since your question refers to stacking gel only. Is the running gel polymerising well? If so, and you use the same chemistry for both gels, than your recipe is wrong. Try to find Hoefer or GEHC Lifesciences electrophoresis booklets. There you have really excellent recipes and instructions how to do not only SDS-PAGE, but some other as well (IEF, PAGE...).   

I also agreed with Mr. Christian. Check the percentage of acrylamide and bisacrylamide, while preparing acrylamide/bisacrylamide solutions. Before preparing polyacrylamide gel, you should keep it at TRIS and other buffers a room  temperature. Use freshly prepared APS and also add optimum TEMED. Better take a capped tube and mix all the buffers required in appropriate quantity as per the percentage of gel add APS and TEMED at last mix it well and then pour it in the casting plate. There must be some remaining buffer left behind in the tube. Keep this casting plate and your capped tube in incubator accelerate polymerisation. Check the tube if the gel is polymerised there, it mean the polymerisation over.

cheers

I wrote: "Is the running gel polymerising well?" Of course, this was a mistake. I should have asked if stacking gel is polymerising well. Sorry for that.

Important ingredients for SDS page are. Acrylamide and Bis acryl amide, TEMED and APS. The polymerazation and solidification of gel is faster depends or APS and TEMED amount added. Increasing amount of APS and TEMED you can reduce time required for polymerization. APS must be freshly prepared each time. 

Concerning the freshness of APS; it is a good idea to prepare aliquots of APS and store them at -20. Consider how many gels you typically pour in a single go, and let that determine the volume which you use to prepare aliquots. 100uL worked very well for me.

if you are following any protocol make sure that you put the right quantity of temed and persulfate or check if there anything wrong with them.  For me it takes 5 minutes to solidified after shaking for fe seconds.,

Room temperature is important, as Dr. Scheckhuber suggests. I remember during cold days that I had to use a heat source near (but not directly on) my gels in order to achieve polymerization in a reasonable amount of time

I have attached the recipe for gel preparation. This may be of help to you.

Moreover bis- acrylamide is missing in the method which has been mentioned by you. 

please try with 10% APS and  30% Acrylamide stock (Acrylamide  29.2 g,N’-N bis methylene Acrylamide 0.8 g, Dissolve to make a final volume of 100ml. Filter and store at 4 degree C in Dark bottles)                  

                  )

YOu have to have Acrylamide an bisacrylamide combo like the others have mentioned; usually it is the APS which has to be freshly prepared. I have stored APS in fridge for a week if I am using it every day. But better fresh.

please prepare APS freshly every time and better to add at the end

APS mus be prepared just before use it.

May be Temed or APS had expired. I have similar problem once before, solved ehen replaced these.

Maybe your acrylamide solution is too old. Use a fresh one .

Try to use a heat source while you are working especially in cold days.

This is my "recipe":

I allways made 2 gels therefore I prepare volume to 2.5 gels.

Resolving gel to 2.5 gels:

Watter milliQ: 4.125 ml, Acrilamide/bisacrilamide 30%: 5 ml, Tris-HCl (1.5 M pH 8.8): 3.125 ml, SDS 10%: 0.125 ml, APS 10% (made just before): 0.125 ml and TEMED 0.005 ml.

Stacking gel to 2.5 gels:

Watter milliQ: 1.75 ml, Acrilamide/bisacrilamide 30%: 0.415 ml, Tris-HCl (0.5 M pH 6.8): 0.315 ml, SDS 10%: 0.025 ml, APS 10% (made just before): 0.025 ml and TEMED 0.0025 ml.

Hate (incubat at 37 ºC) accelerates the polimerization of geles, but I never hate my gels.

I would suggest to check if 30% Acrilamide/bisacrilamide solution is not too old. Had this problem some time ago. As for APS...never had a problem, usually keep my stock frozen, it always works.

Good luck :)

I agree that APS solution should be fresh!

How old is your TEMED? TEMED oxidises when opened and in the past I have had problems with gels polymerising due to an old batch of TEMED. Also, as said above, if your the APS and acrylamide are too old, this will also have an effect on your gels setting.

Yep, I agree with the above posts. Try fresh APS! If it's not the APS it might be the TEMED or acrylamide.

Consider covering your running gel with isopropanol during polymerisation.

Just gently overlay your stacking gel (liquid with ~1-2 ml of Isoprop after pouring it into the casting form, this excludes air and helps polymerisation.

After hardening pour of isoprop and flush with water, remove excess water in the edges with filter paper.

I Agree with the all above posts. Make sure you clean all the plates used for making the gels  with 70% alcohol before you use thoroughly.

 Try to use 30% Bis acrylamide (Please careful while handling).

Make sure you prepare fresh APS everytime while you prepare the gel.

The polymerization reaction will be start once you will add APS and TEMED.

Once you pour your Resolving buffer into the gel cast add isopropanol to give the smooth texture to the gel. 

good luck!!!!! 

Don't forget outgassing of your solutions before pouring the gel.

Had the same problem once. It was the APS in my case. Make a fresh lot 

Just make a fresh APS and add more TEMED than recommended in normal available protocols. Just add around 15-20 ul TEMED to your resolving gel

it is mainly because of difference in the ratio of APS and TEMED that you have used. Also if there is any change in the pH of your buffer that might also contribute to this.

I. if you are not using a fresh prepared APS (or stored at -20ºC), then, use it. If you are, then change the APS batch.

II. You could also change the amount of APS and Temed you are using. I advice, for this volume of mix you are using (aproximately 4mL), between16 and 25µL of APS 25%, no more.

Try u single this composition for 5ml 10% resolving gel

1900uL DW.

1700uL 30% acrylamide mix

1300uL 1.5M Tris HCL pH 8.8

50uL 10% SDS

50uL 10% (w/v)  freshly prepared APS

2-4 uL TEMED ( ammount of TEMED can be adjusted depending on how fast you can manage it to cast) . And if u want to change the concentration of gel from 10-12 or 15 % just make adjustment in the amount of acrylamide and water

Please, see this old, but excellent electrophoresis booklet attached. There you have all information regarding electrophoresis you practically need.

Last time my friend got the same problem like that and finally he realized that he made a wrong percent of APS. Thus, please check your calculation for making APS first!.

my gel prescription:

10% separating  gel:

2,5ml 1,5M Tris HCl pH=8,8 with SDS

3,3 ml 30% acrylamide

4ml water

6% thick. gel:

1,25 ml 0,5M Tris HCl pH=6,8 with SDS

1 ml 30% acrylamide

2,75 ml water

directly before gel preparing add:

• separating gel - 9,8 gel + 75mikrol APS+ 70mikrol TEMED
• thick. gel - 5 ml gel +50 mikrol APS + 50 mikrol TEMED

APS always freshly prepared

Make fresh APS

APS and other solution should be fresh.

Prepare 10%APS freshly and check Ph of tris-Cl, check your TEMED is working properly, try to make 12% SDS.  These things may help you..

All the best..

Room temperature also important for hardening of the gel. In low temperature gels not getting hard some times. Try to keep room temperature around 25 to 28C .  Also check APS and TEMED working properly. 

Get fresh sample of APS which is usually the problem. I trust you have bis acrylamide in your acrylamide solution.

I think APS is the main problem , prepare fresh APS and use

I agree to Christian Q. Scheckhuber's suggestions. I would just like to add one more-although the bottle may not say that ammonium persulfate is light sensitive, put APS in foil wrapped tube following with fresh preparation. 

Referring to Hemangini's suggestion, which I support (wrapping to protect from light which is also demand for acrylamide/bis solution), keep ammonium persulfate crystals out of direct contact with aluminum foil, specially if some microdrop of water is there. I usually measure things putting them to measure on aluminum foil over on the balance. May not  be good idea for APS sometimes.

ya problem might be with APS or old stocks of acrylamide

Dear Rashid,

I have also faced the same problem. I agree with Leonidas. Room temperature is an important factor for polymerization of acrylamide gel. Cold temperature delays the polymerization. 25 °C to 30 °C accelerates polymerization rate. Moreover, use fresh APS as suggested by others.

Hope it will work.

I too agree with Rashid. Check whether your TEMED solution working properly. you may increase the TEMED a little.

Hi,

Use fresh APS and TEMED.

Use saturated butanol for gel overlay as oxidation prevents polymerization of acrylamide.

Let the gel stand and do not disturb it often as it also affects polymerization.

All suggested points are the most possible reason. Be aware of degradation of your starting materials as well.  AMS, TEMED and Bis AA are very vulnerable to chemical decomposition, so check the starting chemicals. 

Other point that may work is using a method to degas the mixture before adding the polymerization trigger. 

Last not the least, if you are using some gel bland or repel solution on the glass plates, they may interfere with casting the gel.

Good luck

some suggestions might help you.

• your temed volume is very low,better increase it,if  you are using old gel procedure means like adding all chemicals separately,then in this case you should add temed volume equal to your final volume.For eg,for 10 ml,then temed should be 10 microlitre.We are doing same thing in our lab.This may be one of the reason for your problem,try it.GoodLuck