i guess, it has to do with the buffers (Tris pH-6.8 and 8.8). please check their pH and their Molarity. make it to the standard and try preparing the reagents fresh and autoclave the tris buffers only. One more thing is after casting the resolving please do not forget to add 1/2 ml of water or isopropanol to prevent oxidation of the gel in contact with air. delay in adding this could also lead to such cases.

hope it was of help to you,

saby

Differences in the saltiness of the samples can cause the saltier ones to spread to the sides, at the expense of neighboring lanes.

I agree with the others.  Your idea of temperature control is correct.  It is important to make the process as isothermic as possible.  Also,  differential pressure caused by gel clamps can contribute to this anomaly.

Michael

Hi there,

In my experience, the presence of urea in one sample provokes its lateral diffusion during migration if neighbouring samples are urea free. I would suggest you to make sure sample composition is roughly the same for the set of samples including protein markers.

dialysis will help you to fix the problem.

Try  to run your sample protein on 12% gel, 10% gel needs proper handling.

If this issue remain, then  try to use freshly prepared reagents. I think this problem will resolved.

Hi Evren,

I agree with the above suggestions.

in my experience, I see "broader" bands when there is a larger amount of protein in the lane.

The lower big red box you present in your gel, in my opinion, is due to not running the gel until the "end"; usually I run the gel so all the sample buffer/front of the gel gets out, so you don't get that line in the end.

The smaller red boxes are just the gel "folding" that is detected by the scanner. i normally use plastic sheets (like this, i don't know the name in english, https://www.google.pt/search?q=mica&espv=2&biw=1224&bih=682&source=lnms&tbm=isch&sa=X&ved=0CAYQ_AUoAWoVChMI5qnRgtDlxwIVxbgUCh1ujw7I#tbm=isch&q=micas+de+plastico&imgrc=2lrZFhevN2giiM%3A), so the gel is flat and that doesn't happen when you take the foto/scan.

Hope this helped!

Hello,

I will check all of your advices and I will try them all as soon as possible. Thank you very much for your concern and advices. I am still open for new ideas and advices until I will try them all. 

I couldn't understand dialysis advice...

When I back to my lab i will check the molarity pH and the procedure. And, What if I use small amount of much APS (Ammonium persulfate) to make gel rigid? Would it cause a problem due to quick polimerization for gel?

I will also try mica to remove gel from glasses.

Next experiment will be running with water cool and I will wait end of the experiment (I usually wait but this time i will observe again.)

And by the way, I am planning to remove gel in a pool that contains tank buffer, with mica plastic. After all advices and experiments I hope we can solve this and want to write experiences here.

Thanks for everyone, I will let you know results and while in this time I will share updates.

Edit: I will ask my supervisor to use %12 gel and if possible I will also try this.

12% gel will resolve your problem, don't use less amount of APS, just use 10% freshly prepared APS

Dear Evren your gels are not so bad!

If are you using SDS-PAGE in Leammli buffer  have a check of the Tris-HCl solutions (6,8 and 8,8),  how is old the acrylamide-bis solution, prepare a fresh APS as already suggested and don't overload the wells.

The gels should not heat up during running, check the watts in your power supply that must not exceed 10 for 2 gels. You can run your gel at room temperature because at 4°C the SDS may precipitate. I suggest also to prepare the gels the day before you will use them

Dear Evren,

please wait for couple hours until the gel matures, polymerization of acrylamide has to be completely finished.

Hello everyone,

Firstly, I would like to thank you for your all advices and sharing. I solved the problem.

1- Gel must be run at cold (about 8-10 C° ) 

2- Gel holder glasses must be separate at in a water pool, thus, gel will be cleaned from SDS and also while separation gel will not have damage because of the vacuum effect. We were trying to separate glasses in streaming water tap and it wasn't wet enough also vacuum effect was damaging the gel. With this way, water will be vacuumed to empty layers and will not damage the gel.

3- Our tank system "Thermo Fisher OWL 8"  and we are preparing gel in a plastic pocket it is not sealed enough while we are loading (liquid form) gel that is why I had "folded gel" at the bottom of gel. Next time, I will use strech film until gel become solid. This way will seal the bottom and it will be well formed I think.

4- These all was performed in %10 SDS Gel, we couldn't change the concentration because of our samples. Gel concentration was made our samples properties.

I hope, this informations will also help other people. Thank you for your concern and generous help.

Best regards.

http://www.thermoscientific.com/content/tfs/en/product/owl-dual-gel-vertical-electrophoresis-systems.html