Fresh APS every time??? Are we talking fresh from dissolving the powder APS? Because if you mean diluting a stock solution or something else, that's not fresh. And indeed, at least 90% of the time, APS is the problem.

A single bubble wouldn't stop your gel from polymerising (although it will mess your gel so don't let it happen). If it's not the APS, then maybe the temperature might be a problem - if it's too cold it will make it difficult for the gel to polymerise. Sometimes people get confused by agarose gels where cold=fast but you need a chemical reaction to occur, and cold makes it difficult for this reaction to start (so basically, if you tried to cool it down you went the wrong way). I'm not talking about heating the gel up ofc, just that if you are feeling kinda chilly in there, your gel might "feel" the same way too. Personally when I'm in a hurry I increase the TEMED concentration.

Lastly, I'm not sure what kind of problem you have with the way gels run, but if it's the dye it could be that you have too much of it in your loading buffer, but I wouldn't care if it doesn't affect your protein samples. If you don't get your samples running evenly, but rather form a "smile" then you are running the gel too fast. (If you care for a "mechanistic" explanation: Running it fast means it overheats, heat escapes to the surrounding air at a different rate from the middle and the edges of the gel, middle and edges end up with different temperature, different temperature means different electrical resistance, which means more current passing through the colder parts, which means the proteins there move faster).

Check if you Ammonium persulphate is new.. Old ones won't work..

Thank you sir for the reply. i have prepared APS fresh eveytime i try casting the gel.... but its a total failure....tried APS and TEMED at different volumes for different % of gels but in vain....

I used to have the same problem and to solve it I increased the concentration of APS (roughly take 1/3 the of the volume of the eppendrof tube + 1 ml H2O) use for the first try then if not working u can take from the fresh prepared APS the double volume........ also u can try different volumes of APS and TEMED for the same gel % until u reach a full polymerization in an appropriate time

hey thank you Makarim Osman... will try that and back to you...hope this solves my problem... can you suggest a book or website where i can get reasons for getting waved, spiked, frown etc dye front while running my SDS-PAGE....

Are you allowing any air to access the gel (bubbles, gaps under the comb) etc? It won't polymerise in the presence of oxygen...

Fresh APS every time??? Are we talking fresh from dissolving the powder APS? Because if you mean diluting a stock solution or something else, that's not fresh. And indeed, at least 90% of the time, APS is the problem.

A single bubble wouldn't stop your gel from polymerising (although it will mess your gel so don't let it happen). If it's not the APS, then maybe the temperature might be a problem - if it's too cold it will make it difficult for the gel to polymerise. Sometimes people get confused by agarose gels where cold=fast but you need a chemical reaction to occur, and cold makes it difficult for this reaction to start (so basically, if you tried to cool it down you went the wrong way). I'm not talking about heating the gel up ofc, just that if you are feeling kinda chilly in there, your gel might "feel" the same way too. Personally when I'm in a hurry I increase the TEMED concentration.

Lastly, I'm not sure what kind of problem you have with the way gels run, but if it's the dye it could be that you have too much of it in your loading buffer, but I wouldn't care if it doesn't affect your protein samples. If you don't get your samples running evenly, but rather form a "smile" then you are running the gel too fast. (If you care for a "mechanistic" explanation: Running it fast means it overheats, heat escapes to the surrounding air at a different rate from the middle and the edges of the gel, middle and edges end up with different temperature, different temperature means different electrical resistance, which means more current passing through the colder parts, which means the proteins there move faster).

Using completely fresh APS each time (that's why u should prepare it in a very small amount each time).. any thing that may affect the running profile mainly caused by the running buffer (pH, ion concentration, temp..)..

Will try to find u some references and feed u back later.

Increase APS or TEMED.

Use fresh APS and new TEMED. (i use 50 uL 10% APS and 10 uL TEMED for 10% Separating Gel)

The temperature is too low, cast at room temperature.

Quality of the acrylamide or bis is poor.

Just adding on. Usually APS powder when dissolved gives out a cackling//popping sound. If not, it most likely is due to APS decomposition in presence of water (*Thus, you might want to get a new bottle). Here is a reference to the article.

http://onlinelibrary.wiley.com/doi/10.1002/bscb.19720810138/abstract

I know this is 7 years too late but I had the same problem this week. My awesome advisors put me on the right track: try using 20% APS instead of the usual 10%. It worked like a charm!

Has anyone tried warming their fresh APS solution? In theory, this should increase rates of polymerization?

Divya!

Thank you! I was having a problem this morning and your answer really helped!

Cheers,

I know this is an old post but reading it gave me some light.

I've been trying to make my gels polimerise for a while now and sometimes they work and sometimes they don't. When they do work, the protein runs are always very different.

Ho Chun Loong 's comment made me realize my APS might be degraded, since I found a crack on the bottle. I'll buy a new one and try it.