Hi, can I ask you if this a gel coloured with comassie and we see the dye front?  I don't see the marker, did the marker migrate well or not? If you checked the gel I am sure you pay attention to the polymerization, but sometimes, even if the gel is uniformly poured, it is non-uniform polymerized.

Thank you Valeria for your answer.

No, it´s not stained with coomassie and the marker migrate well  but sometimes in the lower part of the gel the marker shows diffuse bands.I have never experienced this problem before,I mean in my previous lab;  that´s why I am confused.

Hi Celimene,

Could it be due to a problem of buffers uncompatibility? For isntance, you run the gel in a buffer A and the gel was polymerized in an uncompatible buffer B ... Have you checked this ?

Pr Philippe Urban

Hi, I asked you about the comassie because you said that the high molecular weights bands was ok but not the lower ones, how do you see this, with ponceau? I wolud colour the gel with comassie, and the menbrane with ponceau, after transfer and take a picture of the bands pattern to understand what happen.

I would think about the reagents and the apparatus. Did you coworkers used the same regents? If you just checked the pH and made fresh buffer, you can check if, for example APS or Temed are old or not. As I said before sometimes can happen that you pour your gel without problem but the polymerization is too fast or too slow, in either situation you have a non uniform polymerized gel, in your case the bottom respect the upper part. Did your apparatus is new or old? How you can be sure about the voltage because someone else use the same power suplly without problem? Sorry for all the questions but I think was somenthing about the gel polymerization, if you said that you check all the reagents maybe is something else.

Thank you Pr. Philippe for your answer.

No there is no buffer incompatibility. Could it be that the fact that I overlay my separating gel with distilled water is responsible?

Celimene,

I'm afraid yes !!! It's easy to check though, just overlay them with teh correct buffer are the correct concentration and at teh correct pH.

Philippe

Thanks you Pr. Phillipe. I just started preparing a new one with propanol overlay. Will let you know if it works. I always did with water in my previous lab and never got this problem but I assume that from one lab to another , systems are differents.

Celimene

Thank you Valeria for the answer.

I know the size of the band based on the marker. Actually I think a figured out what the problem is. it might be the separating gel overlay and I will try to solve it and see if I have better results

best

Hello, I need to ask you out of curiosity if you add APS prior to adding TEMED to the gel cocktail ? If yes, how much is the time difference?

Yes Saicharan , I add APS before TEMED and there´s not time between-

This looks very much like a problem between the stacking and separating gel. What % acrylamide are you using for both. We used to use 50% water/50% EtOH to overlay. Do you see a line separating the stacking and separating gels after they polymerize? What do your molecular weight markers look like?

I agree it would be best to see a photo of the entire gel before you run the loading dye off the bottom in order to help diagnose the problem.

Best wishes, 

Ellen

Another possibility is that you might have either loaded too much protein on the gel, or the salt concentration in your samples is too high. How much protein do you load and what is the source? You could try loading less protein (which would also lower the salt concentration), or you could try to do a buffer exchange on your samples to remove the high levels of salt. Good luck!

You may also want to consider if you having enough running buffer in your electrophoresis apparatus. I was having a similar problem and found that it was because the gel was overheating. By simply using a larger volume of buffer to prevent overheating, it vastly improved the outcome.

try H2O-saturated Butanol, (easy to make you just mix butanol and water a wait phase separation, the upper phase is the butanol), 100uL of it loaded on sep gel (mini protean size) is enough. load it from the side and gently. Once separating is polymerase just wash under millipore water before to pour the staking.

Thank you all for your answers.

Actually I load 30 µg of proteins in each well and I will also increase the volume of the running buffer. Do you think that it can be a problem of protein denaturation?